Báo cáo Y học: The processivity and fidelity of DNA synthesis exhibited by the reverse transcriptase of bovine leukemia virus pot

The processivity and fidelity of DNA synthesis exhibitedby the reverse transcriptase of bovine leukemia virus Orna Avidan, Michal Entin Meer, Iris Oz and Amnon Hizi Department of Cell Bi

The processivity and fidelity of DNA synthesis exhibited

by the reverse transcriptase of bovine leukemia virus

Orna Avidan, Michal Entin Meer, Iris Oz and Amnon Hizi

Department of Cell Biology and Histology, Sackler School of Medicine, Tel Aviv University, Tel Aviv, Israel

We have recently expressed in bacteria the enzymatically

active reverse transcriptase (RT) of bovine leukemia virus

(BLV) [Perach, M & Hizi, A (1999) Virology 259, 176–189]

In the present study, we have studied in vitro two features of

the DNA polymerase activity of BLV RT, the processivity of

DNA synthesis and the fidelity of DNA synthesis These

properties were compared with those of the well-studied RTs

of human immunodeficiency virus type 1 (HIV-1) and

murine leukaemia virus (MLV) Both the elongation of the

DNA template and the processivity of DNA synthesis

exhibited by BLV RT are impaired relative to the other two

RTs studied Two parameters of fidelity were studied, the

capacity to incorporate incorrect nucleotides at the 3¢ end of the nascent DNA strand and the ability to extend these 3¢ end mispairs BLV RT shows a fidelity of misinsertion higher than that of HIV-1 RT and lower than that of MLV RT The pattern of mispair elongation by BLV RT suggests that the

in vitroerror proneness of BLV RT is closer to that of HIV-1

RT These fidelity properties are discussed in the context of the various retroviral RTs studied so far

Keywords: bovine leukaemia virus; fidelity; processivity; reverse transcriptase

Bovine leukaemia virus (BLV) is a naturally occurring

exogenous B-cell lymphotropic retrovirus, which is the

aetiological agent of cattle leukosis This disease is

charac-terized by an initial persistent lymphocytosis, which is

followed by the occurrence of clonal lymphoid B-cell

tumours after a long latency period [1] This virus is related

to human T-cell leukemia viruses type I and type II (HTLV-I

and HTLV-II, respectively), forming a subfamily of

trans-activating retroviruses [2] The genomes of these complex

retroviruses have close to their 3¢ ends the regulatory genes

taxand rex and the presence of both Tax and Rex proteins,

encoded by these genes, is required for viral replication

These viruses also show nucleotide sequence similarities,

although BLV and HTLVs do not infect the same cell types,

because they probably bind different cell receptors [2–4]

The process of reverse transcription is the major early

intracellular event critical to the life cycle of all retroviruses

The synthesis of the proviral DNA is catalysed entirely by

the reverse transcriptase (RT) The plus-strand viral RNA is

copied by the RNA-dependent DNA polymerase activity of

RT, producing RNA/DNA hybrids The intrinsic ribo-nuclease H (RNase H) activity of RT specifically hydrolyses the RNA in these heteroduplexes Finally, the plus-sense DNA strand is synthesized by copying of the minus-sense DNA strand by the DNA-dependent DNA polymerase (DDDP) activity of RT [2,5] As RT is a preferred target for the development of viral inhibitors as antiretroviral drugs, the structural and catalytic properties of RTs have been the focus of many recent studies, including three-dimensional crystal studies [6–9] A major effort was devoted to the research of the RTs of the human immunodeficiency viruses type 1 and type 2 (HIV-1 and HIV-2, respectively), which are responsible for acquired immunodeficiency syndrome (AIDS); most of the anti-AIDS drugs approved so far for the treatment of AIDS are inhibitors of the viral RT Due to the rapid emergence of drug-resistant HIV RT variants, the development of novel potent and specific inhibitors of HIV RTs is still a principal objective in the chemotherapy of AIDS [2,10,11] Targeted drug designs rely on a better understanding of the structure and function of retroviral

RT Therefore, the investigation of RT of other retroviruses should expand our understanding of the catalytic properties

of these closely related proteins

We have recently expressed the recombinant RT of BLV

in bacteria The gene encoding the RT was designed to start

at its 5¢ end next to the last codon of the mature viral protease; namely, the amino terminus of the RT matches the last 26 codons of the pro gene and is encoded by the pol reading frame [12] BLV RT was purified and studied biochemically: it exhibits all activities typical of RTs, i.e both RNA- and DNA-dependent DNA polymerases and RNase H activity Unlike most RTs, the BLV RT is enzymatically active as a monomer even after binding a DNA substrate The enzyme shows a preference for Mg2+ over Mn2+ in both its DNA polymerase and RNase H activities BLV RT was shown to have a relatively low

Correspondence to Amnon Hizi, Department of Cell Biology and

Histology, Sackler School of Medicine, Tel Aviv University, Tel Aviv

69978, Israel Fax: +972 3 6407432, Tel.: + 972 3 640 9974,

E-mail: ahizy@post.tau.ac.il

Abbreviations: BLV, bovine leukemia virus; HTLV, human T-cell

leukaemia virus; HIV, human immunodeficiency virus; MLV, murine

leukaemia virus, MMTV, mouse mammary tumour virus; AMV,

avian myeloblastosis virus; EIAV, equine infectious anaemia virus;

AIDS, acquired immunodeficiency syndrome; F ins , frequency of

insertion; F ext , frequency of extension; DDDP, DNA-dependent DNA

polymerase; RNase H, ribonuclease H.

Note: M E Meer and O Avidan contributed equally to the research

described in this manuscript The results presented are in partial

ful-filment of a PhD thesis (M.E.M.) at Tel Aviv University.

(Received 6 August 2001, revised 14 November 2001, accepted 3

December 2001)

sensitivity to nucleoside triphosphate analogues, known to

be potent inhibitors of other RTs such as that of HIV [12]

In the present study we have extended the investigation of

BLV RT by an in vitro analysis of the processivity and the

fidelity of DNA synthesis (namely, the ability of BLV RT to

misincorporate nucleotides at the 3¢ end of the growing

DNA strand and the further extending of the preformed

mismatches)

M A T E R I A L S A N D M E T H O D S

Recombinant RTs and DNA polymerase activities

Monomeric BLV RT was expressed in bacteria and purified

to homogeneity after modifying the method published

recently [12] These modifications were as follows: (a) the

pUC12N6H BLV RT-transformed Escherichia coli were

grown in Terrific Broth (without glycerol) instead of

NZYM medium; (b) the carboxymethyl Sepharose column

buffer was at pH 6.5 (instead of pH 7.0); (c) after the

purification had been carried out the BLV RT was further

concentrated in an Amicon Centriprep 30 concentrator

Recombinant heterodimeric HIV-1 RT was expressed in

bacteria as described [13] Recombinant murine leukaemia

virus (MLV) RT was also expressed in E coli [14] The

recombinant proteins containing six histidines at their

amino termini were purified as described above for BLV

RT, except for the fact that all buffers used to purify MLV

RT included 0.2% (v/v) Triton X-100 (instead of 0.1%)

The DNA polymerase activities were assayed as described

previously [15] One unit of activity was defined as the

amount of enzyme that catalyses the incorporation of

1 pmol dNTP into activated DNA (that served as the

template-primer) in 30 min at 37°C, under the assay

conditions Similar BLV, HIV-1 or MLV RT DNA

polymerase activities were used in all experiments described,

using 0.1–0.5 lg RT protein (according to the specific

activities of the different enzymes)

Template primers

For the experiments of DNA primer extension and

processivity, we used single-stranded circular /X174am3

DNA (from New England Biolabs) as the DNA template,

which was primed with a 15-residue synthetic primer

(5¢-AAAGCGAGGGTATCC-3¢) that hybridizes at

posi-tions 588–602 of the /X174am3 DNA The synthetic

template-primers used for the experiments of misinsertion

and preformed mispair extension are shown in Figs 2 and 3

For analysis of site-specific nucleotide misinsertion, a

synthetic 50-residue template (with a sequence derived from

nucleotides 565–614 of /X174am3 DNA) was primed with

the same 15-residue oligonucleotide used for extension and

processivity This primer hybridizes to the sequence at

positions 24–38 (in the 5¢ fi 3¢ direction) of the 50-residue

template DNA (Fig 2) For the extension of DNA from a

mispaired terminus, the set of template-primers used is

composed of the same 50-residue oligonucleotide template

used for the misinsertion studies (Fig 3), primed with a set

of 16-residue oligonucleotides (that hybridize to the

nucleo-tides at positions 23–38 of the template) Four versions of

16-residue primers were used, each differing from the other

only at its 3¢-terminal nucleotide (Fig 3) All primers used in

this study were labelled at their 5¢ ends with c[32P]ATP (using T4 polynucleotide kinase) and were annealed to the templates with a twofold molar excess of each template over its primer as described previously [16]

DNA polymerization and processivity experiments The reactions were conducted in a final volume of 12.5 lL 6.6 mM Tris/HCl, 4 mM dithiothreitol, 24 lgÆmL)1 BSA,

6 mM MgCl2(for BLV and HIV-1 RTs) or 1 mMMnCl2 (for MLV RT), final pH 8.0, supplemented by the /X174am3 template-primer at a final concentration of

30 lgÆmL)1 For processivity studies, the BLV, HIV-1 and MLV RTs, at equal DNA polymerase activities, were incubated with the annealed template primer for 5 min at

30°C In all polymerization experiments shown we used 0.3–2 pmol RT per reaction (depending on activity) and

Fig 1 DNA primer-extension and processivity of DNA synthesis exhibited by BLV, HIV-1 and MLV RTs All reactions were performed with the 15-nucleotide synthetic 5¢ end-labelled oligonucleotide primer and a twofold excess of the template single-stranded circular /X174am3 phage DNA The sequence of the primer and the experi-mental details are described in Materials and methods The symbols for the DNA synthesis experiments are as follows: (–) DNA extension performed with no DNA trap; (+) DNA extension experiments conducted in the presence of unlabeled DNA trap Molecular mass markers are HinfI-cleaved dephosphorylated double-stranded /X174am3 DNA fragments (Promega) labelled with [c- 32 P]ATP at the 5¢ ends by polynucleotide kinase.

 2.5 pmol of the template primer The reaction mixtures

were divided into two, without or with a DNA trap of a

large excess of unlabelled activated herring sperm DNA,

at a final concentration of 0.6 mgÆmL)1 (prepared as

described previously [15]) All reactions were initiated

immediately afterwards by adding the four dNTPs, each at

a final concentration of 20 lM, followed by incubation at

37°C for 30 min The reactions were stopped by adding

an equal volume of formamide dye mix, denatured at

100°C for 3 min, cooled on ice and analysed by

electrophoresis through 8% polyacrylamide/urea

sequenc-ing gels in 90 mM Tris/borate, 2 mM EDTA pH 8.0, as

described previously [17] The extension products were

quantified by densitometric scanning of gel

autoradio-grams and the amounts of primer extended were

calcu-lated

Fidelity of DNA synthesis

For site-specific nucleotide misinsertion, we assayed dNTP

incorporation opposite to the A residue at position 23 of

the template as described [16,18] (see also Fig 2) The

32P-5¢-end-labelled 15-residue primer was extended in the

presence of increasing concentrations of either 0–1 lM of the correct dNTP (dTTP) or 0–1 mMeach of the incorrect dNTPs (dATP, dCTP or dGTP) All dNTPs used were of the highest purity available (Pharmacia) with no detectable traces of contamination by other dNTPs For mispair extension (Fig 3), elongation of 32P-5¢-end-labelled 16-nucleotide primers was measured with increasing con-centrations of dATP as the only dNTP present (0–1 mM range for the mispaired AÆA, AÆC or AÆG termini or a 0–1 lM range for the AÆT correct terminus) [16,18] Reactions for all kinetic analyses contained 14 mM Tris/ HCl pH 8.0, 4 mM dithiothreitol, 4 mM MgCl2 and

24 lgÆmL)1BSA The reactions were incubated at 37°C for either 2 min (for the correct incorporation or correctly matched DNA elongation), or 5 min (for misincorporation

or extension of formed mismatched DNA) Kinetic reactions were performed with an 10-fold molar excess

of template-primer over BLV RT to ensure steady-state kinetics in the linear range All reaction products were analysed by electrophoresis through 14% polyacrylamide/ urea in Tris/borate and EDTA sequencing gels, and band intensities were quantified as described above This allowed the calculation of reaction velocities, i.e the amount of

Table 1 Quantitative analysis of DNA primer-extension and relative processivity of DNA synthesis The radioactivity in the DNA bands in all polynucleotide length ranges were summed and then the values obtained were divided by the sums of all extended and unextended primers (detected

in the phosphoimaging scanning of the gels as shown in Fig 1) The values given are the extended primers in each product length range expressed as percentages of the total amounts (all extended and unextended primers) of the DNA products The calculations were conducted separately for gel lanes of reactions carried out in the absence or presence of an excess of the unlabeled DNA trap (see Materials and methods) The overall extensions

in the presence of the DNA trap, divided by the comparable figures obtained with no trap present, yielded the relative processivity values expressed

as percentages (see text) The values are the menas calculated from two independent experiments (one of which is shown in Fig 1) and the variations were usually < 15%.

Product length

(nucleotides)

Without trap With trap Without trap With trap Without trap With trap

Table 2 Quantitative analysis of DNA synthesis and processivity after correcting for the relative length of the DNA products The data shown were derived from the same two independent experiments as in Table 1 Here, the data were evaluated after correcting for the mean lengths of the DNA primers extended by the three RTs under the assay conditions used The correction for the actual amount of dNTP incorporation for a given DNA product was achieved by multiplying the radioactivity in each 5¢ end-labelled polynucleotide product length class by the median of the number of nucleotides added in each range (i.e 17 nucleotides for the 16–50 nucleotide range, 42 nucleotides for the 51–100 nucleotide range, 92 nucleotides for the 101–200 nucleotide range and 342 nucleotides for the 200–700 nucleotide range) After introducing these factors, all values are expressed (as in Table 1) as percentages of the total amounts of all primers extended in each length class The values shown are the means calculated from the same two independent experiments as in Table 1.

Product length

(nucleotides)

Without trap With trap Without trap With trap Without trap With trap

total 32P-labelled primer extended per minute in the

conditions used The Vmaxand Kmvalues were calculated

from the double-reciprocal linear plots of velocity vs

dNTP concentrations [16,18]

R E S U L T S

We have analysed in vitro two basic properties of DNA

synthesis by BLV RT, i.e processivity and fidelity, both in

comparison with the well-studied RTs of HIV-1

(represent-ing a low fidelity RT from the lentivirus subfamily of

retroviruses) and of MLV (representing a relatively high

fidelity RT from the mammalian type C retroviruses)

[16,19–21] The assays were performed with template-primers already used in our laboratory with other RTs, allowing comparison of information Similar to all RTs studied so far, BLV RT was found to lack a 3¢ fi 5¢ exonuclease (proofreading) activity (data not shown), thereby permitting direct kinetic analysis of primer-exten-sion Previous data show that BLV RT, like HIV-1 RT, prefers Mg2+over Mn2+[12] Therefore, all assays carried out with these RTs were performed in the presence of Mg2+ ions For MLV RT, we have evidence that overall extension

of primers by this RT in the presence of Mn2+is far better than with Mg2+, whereas the fidelity of DNA synthesis by MLV RT (both misinsertion and mispair extension; see below) is similar with Mg2+or Mn2+(unpublished data)

DNA synthesis under processive and nonprocessive conditions

The processivity of a DNA or RNA polymerase is directly proportional to the length of the nascent polymeric products formed before the enzyme molecules dissociate from these product molecules and rebind the same or other template-primer molecules [17,22] The extent of product elongation

in one cycle of synthesis (before the polymerase disassociates from the growing strand) may depend on kinetic parameters that affect binding, single nucleotide addition, translocation, pausing, etc It is apparent that retroviral RTs are far from performing totally processive events (where the entire template molecule is copied as a consequence of a single binding event of the enzyme) [17] Therefore, we have tested the processivity of the BLV RT in comparison with the two well-studied RTs of HIV-1 and MLV

In the primer-extension assay, described in Fig 1, we used the heteropolymeric single-stranded /X174am3 DNA

Fig 2 The pattern of DNA mispair formation by BLV, HIV-1 and MLV RTs The synthetic 50-nucleotide template was annealed to the 32

P-5¢-end-labelled primer The primer was extended with equal DNA polymerase activities of either BLV RT, HIV-1 RT or MLV RT in the presence of 1 m M

of a single incorrect dNTP (i.e C, G, or A) or 1 l M of the correct dNTP (dTTP) as described in Materials and methods The level of misinsertion is apparent from the elongation of the primer in the presence of the incorrect dNTP relative to that in the presence of dTTP.

Table 3 Kinetic parameters for site-specific misincorporation by BLV

RT The 15-residue c ) 32

P-5¢-end-labelled primer was hybridized to a fourfold molar excess of the 50-residue template derived from the

sequence of nucleotides 565–614 of /X174am3 DNA (Fig 2) In each

set of the kinetic experiments, the template-primer was incubated with

BLV RT in the presence of increasing concentrations of a single dNTP.

The oligonucleotide products were analysed and described in Materials

and methods The apparent K m and V max values were determined from

at least two independent experiments performed as described in

Materials and methods and in the text and the variations were usually

< 20% The values of relative frequency of insertion (F ins ) were

cal-culated as described in the text.

Pair or mispairs

V max

(%Æmin)1) F ins

as the template, which is annealed to a synthetic 5¢

end-labelled primer The extension of the primer by the RTs

was carried out in the absence or presence of a DNA trap,

added to the reaction mixture after the RT is given the

opportunity to bind the template-primer and before

polymerization starts (see Materials and methods) As

the trap is added in a vast excess, only prebound RT

molecules are allowed to extend the labelled primer This

restricts the extension reaction to only one round of

synthesis, hence once RT falls off, it binds the trap and is

not capable of performing further rounds of extending the

labelled primer As expected, all three RTs produce longer

DNA products when multiple rounds of synthesis are

allowed All RTs used have been calibrated to have the

same DDDP activity using activated DNA as the substrate

(see Materials and methods) Yet, the extent of elongation

obtained with BLV RT with no trap present is

substan-tially lower than that with HIV-1 RT and MLV RT Most

products generated by BLV RT are up to  150

nucleo-nucleotides in length, whereas for the other two RTs the

majority of the products are substantially longer than

200 nucleotides The primer-extension labelled products

were quantified and the extent of elongation was calculated

by two methods In the first, we calculated the amount of

product as a percentage of the total radioactivity detected

(Table 1) Both HIV-1 RT and MLV RT show, with no

trap present, overall extensions of 73–76% which is

significantly higher than that of BLV RT (61%) The

majority of the products of the former two RTs (28–35%)

are longer than 200 nt, whereas only 6% of the products

synthesized by BLV RT are longer than 200 nt These

differences are more remarkable after quantifying the

products generated by introducing a correction of the lengths of the polynucleotides synthesized (Table 2) In this method the average lengths of products was taken into account in the calculation, as by being 5¢ end-labelled all oligonucleotides have the same level of label per molecule, irrespective of their lengths This method corrects for the actual amount of dNTPs incorporated per given product The figures calculated by this second method show also that the overall extension of BLV RT is significantly lower than the extension calculated for the other RT studied (68% for the BLV RT and 88% for HIV-1 and MLV RTs)

As expected, when a DNA trap is present and only one round of DNA synthesis is permitted, all RTs synthesize less, as well as shorter, product when compared with multiple-round synthesis (Fig 1) The analysis of the processivity of DNA synthesis in the presence of a DNA trap suggests that BLV RT has a processivity that is substantially different from that of HIV-1 and MLV RTs It

is apparent that BLV RT produces very short products, most of them < 30 nucleotides in length In comparison, HIV-1 RT synthesizes products that are not substantially different in their length from those generated when multiple rounds of synthesis were allowed MLV RT synthesizes, in the presence of the trap DNA, products that are shorter than those produced without a trap (but longer than those generated by BLV RT) The quantitative analysis of the relative processivity depends on the method of calculation When the overall extensions were calculated by the first method outlined above (Table 1) MLV RT shows a superb processivity of almost 100%, whereas BLV RT has substantially lower processivity (54%) which is somewhat

Fig 3 The pattern of mispair extension displayed by the purified RTs of BLV, HIV-1 and MLV The32P-5¢-end-labelled 16-nucleotide primers were hybridized to the 50-nucleotide template, producing duplexes with 3¢-terminal preformed mismatches, where N at the 3¢ end of each represents the incorrect nucleotide (A, C or G) or the correct on (T) The primers were extended with equal DNA polymerase activities of BLV RT, MLV RT, or HIV-1 RT (as described in the text and in Materials and methods) in the presence of either 1 m M dATP (when the mispaired template-primers were elongated) or 1 l M dATP (in the case where the AÆT paired substrate was extended).

higher than that of HIV-1 RT (46%) However, by

calculating the level of extension after correcting for the

lengths of the products synthesized, the data obtained is

substantially different (Table 2) HIV-1 and MLV RTs

exhibit relative processivity values, which are practically

identical ( 64%) whereas BLV RT shows a much lower

processivity of 24%

The fidelity of DNA synthesis

All our previous studies with a variety of retroviral RTs

have shown that the parameters for fidelity of DNA

synthesis in vitro (i.e 3¢ end misinsertion and the extension

of the performed 3¢ end mispaired primers) depend

primar-ily on the sequences of nucleic acids copied, rather than

whether DNA or RNA templates were copied [16,18,20,23]

Subsequently, in the present study we have analysed DNA

templates as representing both DNA and RNA substrates

The formation of 3¢ mispaired DNA To study the fidelity

of misinsertion, we used an assay system that measures the

standing-start reaction of 3¢ end misinsertion This is

achieved by following the misincorporation of incorrect

dNTPs opposite the template A nucleotide, which

corre-sponds to position 23 in the 50-nucleotide template used, in

comparison with the incorporation of dTTP (see Fig 2 and

Materials and methods) The elongation of the32

P-5¢-end-labelled 15-nucleotide primer was performed with either

1 lMof the correct dNTP (dTTP) or 1 mMof each of the

incorrect dNTPs Fig 2 shows the gel analysis of the

elongation products with the correct or incorrect dNTPs It

is apparent that the general pattern of primer extension

obtained with BLV RT is quite similar to this with HIV-1

RT There is an elongation of one nucleotide in the presence

of 1 lM dTTP with no significant further extension The

highest extent of misincorporation is observed with dCTP,

forming CÆA mispairs, which are elongated further creating,

in the case of BLV RT, CÆT mispairs followed by the correct

pairs CÆG (18 nt) In comparison, HIV-1 RT is capable of

elongating further the 18-nucleotide primers to 19

nucleo-tides (with a CÆT mispair at the 3¢ end) With both BLV and HIV-1 RTs, the extent of mispair formation with dGTP and dATP (forming GÆA and AÆA mispairs, respectively) is lower than with dCTP MLV RT shows, on the other hand,

a substantially lower level of misincorporation relative to the other two RTs studied The only significant misincor-poration by MLV RT is apparent with dCTP, forming CÆA mispairs, with no significant further elongation of the 16-mer products with this mispair at its 3¢ end

To quantify the capacity of BLV RT to form 3¢ end mispairs, four separate sets of primer-extension reactions were carried out and analysed In each case, we used increasing concentrations of a single dNTP, thereby determining the standing-start rate of synthesis of the correct pair vs the three possible mispairs We used a range of dNTP concentrations always below 1 mM (to obey steady-state kinetic conditions) and calculated the radioactivity in gel bands relative to the total amounts of primer present (both the unextended and the extended ones) The rates of misincorporation (V ¼ percentage of primers elongated per minute) were calculated as a function of dNTP concentrations, as described in Mate-rials and methods The apparent Kmand Vmaxvalues for each dNTP were all derived from the double-reciprocal curves of the initial velocities of primer extension vs the substrate concentrations (data not shown) and are given

in Table 3 To calculate the frequencies of misinsertions (Fins values) we used the method used previously [16,18,19]:

Fins ¼ ðVmax=KmÞw

ðVmax=KmÞR

where (w) denotes the incorrect nucleotide (dATP, dCTP

or dGTP) and (R) is dTTP As expected from the pattern of primer extensions (Fig 2), the highest Finsvalues calculated for BLV RT is for dCTP (1/11 600, see Table 3), whereas, the formation of AÆA mispairs is very rare (Fins 1/

300 000) and the value calculated for dGTP incorporation is slightly higher (1/62 500) The parallel Finsvalues calculated

by us previously in the same assay system for HIV-1 RT were: 1/3460–1/9000, 1/32 250–1/41 500 and 1/52 200–1/

75 000; and for MLV RT: 1/25 000, < 1/300 000 and

< 1/300 000, all for the formation of AÆC, AÆG, and AÆA mispairs, respectively [16,20]

Extension of preformed 3¢ end mispaired DNA Misin-sertion by itself is not sufficient to create stable site-specific mutations, unless the terminally mispaired DNA is further extended, leading to the fixation of the mistaken sequence Therefore, the efficiency of extending 3¢ preformed mis-matched primers is an essential factor in determining the fidelity of DNA synthesis exhibited by different polyme-rases We have evaluated the ability of BLV RT to extend preformed 3¢ end mispaired 16-residue primers (AÆA, AÆC, AÆG) by analysing the extension of these primers during DNA polymerization in the presence of the next comple-mentary dATP (as the only dNTP present) These standing-start reactions were performed in comparison to HIV-1 RT and MLV RT analysed with the same mispair extension reactions The gel analysis of the extension products shown

in Fig 3 shows that BLV RT is capable of elongating all

Table 4 The kinetics of the extension of 3¢ end matched or preformed

mismatched primer termini by BLV RT The 32P-5¢-end-labelled

16-nucleotide primers were hybridized to a 50-nucleotide template

derived from the sequence of nucleotides 565–614 of /X174am3 DNA,

producing duplexes with a 3¢-terminal paired (AÆT) or mismatched

(AÆC, AÆG or AÆA) primers (Fig 3) Each template-primer was

incu-bated with BLV RT in the presence of increasing concentrations of

dATP The products were analysed as described in the text The

apparent K m and V max values were the means calculated from at least

two independent experiments and the variations were usually < 15%.

The relative frequency F ext values are the ratio of the rate constants

(V max /K m ) for the mispair divided by the ratio of the corresponding

constants for the paired AÆT terminus.

Pair or mispairs terminus K m (l M )

V max

(%Æmin)1) F ext

mispairs to roughly the same extent In comparison, HIV-1

RT shows a substantial preference in extending the AÆC

mispairs over the AÆA and AÆG mispairs MLV RT shows

the same preference in extending the mispairs (AÆC >

AÆA > AÆG) although the extent of elongating these

mispairs is significantly lower than the extensions observed

with HIV-1 RT

To study the kinetics we followed primer elongation as a

function of increasing concentrations of dATP as the only

dNTP present (Table 4) The ratios of all extended products

were calculated relative to the total amount of the primers as

a function of dATP concentration The relative extension

frequency (Fext) values are defined as apparent Vmax/Km

values, calculated for the formed mismatches, divided by the

corresponding Vmax/Km values obtained for the correctly

paired terminus (AÆT) The results show that the apparent

Kmvalues for the extension of all three studied mispairs by

BLV RT are similar As expected, the Vmaxvalue calculated

for the corrected paired terminus is higher than those values

determined for the mispaired termini Also expected is the

finding that this RT exhibits Kmvalues for the extension of

the AÆA, AÆC and AÆG mismatches that are much higher

than the comparable value calculated for the correct AÆT

pair As the extension of all three mispairs is about the same

(Fig 3) it is not surprising that the relative extension

frequencies calculated for all mispairs are quite similar,

ranging from 1/3400 (for the AÆG terminus) to 1/5100 (for

the AÆC mispair) On the other hand the Fext values

calculated previously in the same assay system were for

HIV-1 RT, 1/17 500–52 000, 1/3900–9200 and 1/35 000–

45,000, for the formation of AÆA, AÆC, and AÆG mispairs,

respectively [16,20]

D I S C U S S I O N

Polymerases are processive, i.e they can attach to the

polymeric substrates and perform polymerization cycles

without intervening dissociations [21,24] A total

proces-sivity of synthesis of either DNA or RNA is accomplished

when the entire DNA or RNA template is copied as a

consequence of only one polymerase-binding event

Previ-ous studies with variPrevi-ous RTs have shown that the enzyme

is not highly processive while synthesizing DNA

[17,18,25,26]

The primer-extension and processivity of DNA

synthe-sis experiments shown in Fig 1 indicate that these features

of BLV RT are significantly different than those of both

HIV-1 RT and MLV RT The data were quantified by

two methods (Tables 1 and 2) It is apparent that BLV

RT has a processivity substantially lower than that of the

two other RTs studied Even without an excess of

unlabelled trap DNA, BLV RT is not capable of

synthesizing significant amounts of product DNA longer

than  120 nucleotides, with strong pausings between 90

and 120 nucleotides In comparison, HIV-1 and MLV

RTs synthesize a relatively large amount of longer product

DNA molecules of 200–700 nucleotides in length, and the

majority of the products are in this length range This

difference between BLV RT and the two other RTs

suggests that BLV RT has weaker binding to the DNA

substrate than the other RTs studied It might also be that

the premature pausings observed with BLV RT are

sequence-dependent It will be of interest to study other

sequences than those used here to identify any unique sequences that cannot be copied easily by BLV RT The processivity experiment was conducted with a large excess of trap DNA to prevent rebinding of RT molecules

to the nascent DNA The extent of DNA synthesis with BLV RT is low and most products are very short (Fig 1 and Tables 1 and 2) This shows that these products, generated under single-cycle conditions were synthesized by those BLV RT molecules that were bound to the DNA before the addition of the trapping agent (and were dissociated from the growing chain after incorporating only few nucleotides) suggesting a poor processivity of this RT The pattern of the processivity seen with HIV-1 RT is entirely different Despite exhibiting a moderate processiv-ity, the distribution of the elongation products in the presence of the trap DNA is very similar to that seen in its absence (although, as expected, the total amount of product generated with the trap is lower, only 46–64% of those synthesized without the trap) This phenomenon might suggest that those HIV-1 RT molecules that can withstand dissociation are capable of completing the synthesis (or show a high ÔpersistenceÕ of elongation without further dissociation) MLV RT shows a behaviour that is interme-diate between the apparent features of BLV RT and HIV-1

RT The products formed in the presence of the trap DNA are substantially shorter than those synthesized with no trap present (though they are much longer than those synthe-sized by BLV RT with the trap DNA) The variations observed in the experiment shown in Fig 1 necessitated the use of the two quantification methods, summarized in Tables 1 and 2 BLV RT shows an overall processivity of DNA synthesis, which is significantly lower than the values calculated for both HIV-1 and MLV RTs (see Table 2) Yet, based on the amount of primers extended in the processivity experiments, BLV RT is capable of extending about the same amount of primers as HIV-1 RT ( 50%), despite the very significant differences in the ÔpersistenceÕ of elongation (see Fig 1 and Table 1) MLV RT is capable

of extending many more primer molecules (showing a value

of almost 100% of relative processivity) It is possible that these results may vary slightly depending of the sequence of the DNA copied and the conditions used in the experiments None of the RTs studied so far have any 3¢ fi 5¢ proofreading exonuclease activities, thus, making RTs more error prone than other DNA polymerases with this activity [5,16,18,27,28] Yet, a comparison of the overall fidelity of DNA synthesis exhibited by RTs from different retroviruses reveals significant differences among them It was reported that the RTs of HIV-1 and HIV-2 are relatively more error prone than other RTs, such as those of avian myeloblastosis virus (AMV) or MLV [19,20,23,29,30], explaining the extensive genetic heterogeneity of both HIV-1 and HIV-2, which affects viral pathogenesis, the rapid emergence of drug-resistant variants and, hence, the progression of AIDS [2,10,11,31] We have also found that the relatively low fidelity of DNA synthesis exhibited by HIV RTs is shared

by the RT of equine infectious anaemia virus (EIAV), which belongs to the lentivirus subfamily of retroviruses [16] In general, the fidelity of DNA polymerases results from the combination of nucleotide insertion and extension (in addition to the presence or absence of proofreading activities) Base substitution mutations during reverse transcription can arise from the incorporation of a

noncomplementary nucleotide at the 3¢ end of the nascent

DNA strand, followed by an extension of the preformed

mispair [32,33] Therefore, using parameters of both the

capacity to misincorporate an incorrect nucleotide and the

ability to extend the preformed 3¢ mispairs, it was suggested

that the overall rates of the in vitro error proneness in the

RTs studied is as follows: lentiviral RTs > AMV

RT > MLV RT [16,19,29,30,34] A more recent study

carried out with the RT of mouse mammary tumour virus

(MMTV) has shown some deviation between the efficiency

of misincorporating an incorrect nucleotide and the ability

to elongate such a mispaired DNA [18]

We have studied the error proneness of BLV RT using

the defined template-primers and steady-state kinetics

methods used previously in our laboratory to study various

RTs [16,18–20,23,29,34] The misinsertion frequencies

observed with BLV RT show that the specificity of

mismatch formation is AÆT > AÆC > AÆG > AÆA,

com-patible with the pattern observed with the other RTs [16,18–

20,23,29] This misinsertion is affected by a major increase in

the Kmvalues and a less significant reduction in the Vmax

values The Finsvalues are somewhat different than those

observed previously with HIV-1, HIV-2 and EIAV RTs

The fidelity of misincorporation of MLV RT is substantially

higher than both BLV and lentiviral RTs (Fig 2) and

[19,23] Therefore, the overall order of error proneness of

the retroviral RTs studied, based on the site-specific

misincorporation experiment, is lentiviral RTs > BLV

RT MMTV RT > AMV RT > MLV RT

As to the capacity of BLV RT to extend preformed

mispairs, it is apparent from Fig 3 and Table 4 that BLV

RT extends all mismatches studied (i.e AÆA, AÆC, and AÆG)

to approximately the same extent The enzyme can extend

the mispairs by only one correct nucleotide (A) with no

further extension by misincorporating A opposite to G This

is in contrast with the pattern of elongation observed here

with HIV-1 and MLV RTs (Fig 3) and previously by these

RTs and the RTs of HIV-2, EIAV, MMTV and AMV

[16,18,19,23,29] With all other RTs the efficiency of

preformed mispair extension with the same mispairs was

found always to be in the order AÆC > AÆA P AÆG

Moreover, all RTs except for BLV RT were capable of

extending the AÆC mispair beyond the addition of only one

A This indicates that, under the assay conditions used, all

other RTs can incorporate A opposite to G at position 18

This is true even for MLV RT which has the highest fidelity

of all RTs studied The steady-state kinetics analysis of the

mispair extension by BLV RT shows that the Vmaxand the

Kmvalues are relatively close for all mismatched substrates

(Table 4) Moreover, major discrimination can be attributed

to the relatively large Kmdifferences governing the extension

of matched vs mismatched base pairs, with much smaller

differences in the Vmax values The high frequency of

extending the studied mispairs by BLV RT, relative to our

previous results, puts this RT on the top of the list with

lentiviral RTs in the in vitro error proneness of RTs in the

following order: BLV RT lentiviral RTs > AMV

RT > MMTV RT > MLV RT However, the fact that

the mispaired DNA can be extended by BLV RT by only

one nucleotide beyond the mismatched 3¢ end may explain,

at least in part, why virions of BLV grown in culture show

a relatively low mutation rate per replication cycle [35]

If extension of mispairs stops after the addition of one

nucleotide also in vivo, there will not be synthesis of full-length mutated DNA and the overall fidelity will be relatively high This may also explain the observed in vitro reduced processivity of BLV RT Obviously, other viral and cellular factors may also contribute to the result reported for virions

In summary, BLV RT shows a significantly low proces-sivity of DNA synthesis together with a low fidelity, making BLV RT unique among retroviral RTs It had been suggested already for mutants of HIV-1 RT that there is

an inverse correlation between the fidelity and processivity

of DNA synthesis (i.e that the enhanced fidelity of misinsertion and mispair extension is associated with a reduced processivity [36]) The results with BLV RT in the present study as well as with other mutants of HIV-1 RT [17] do not support this theory

A C K N O W L E D G E M E N T

We thank H Berman for typing the manuscript.

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