Ver-Subunits Polymerase Localization and Function mass in kD Subunit Function DNA polymerase Nuclear; initiation of nuclear DNA replication DNA polymerase Nuclear; principal polymerase
Trang 1of proteins that interact with Dbp11, leads to the recruitment of DNA polymerase to
the replication origins (The protein acronyms refer to DNA polymerase-binding
[Dpb] and proteins encoded by genes that give a lethal phenotype when mutated in
Dpb11 mutant strains of yeast [Sld, or synthetic-lethal with Dpb11] Collectively,
these proteins are referred to as the “11-3-2 complex”) The actions of S-CDK and
Cdc7-Dbf4 trigger bidirectional DNA replication from each origin, with the two
di-verging MCM complexes serving as helicases Each helicase unwinds the duplex
DNA to provide single-stranded templates for the DNA polymerases that follow
Proteins of the Prereplication Complex Are AAA ⴙ ATPase Family
Members
Cdc6, the Orc proteins, and MCM proteins are members of the AAAⴙ ATPase
family, a group of proteins characterized by sequence and structural homology,
ATPase activity, and a general function as molecular chaperones The binding of
both ORC and Cdc6 to chromatin in the process of pre-RC assembly is
ATP-depen-dent (Figure 28.12) ORC with ATP bound can bind to origins of replication and
recruit Cdc6 and Cdt1 to form a pre-RC in which both ORC and Cdc6 have bound
ATP Cdc6 is required for the recruitment of the MCM proteins, specifically, a
com-plex of MCM2–7 with ATP-dependent helicase activity To establish the pre-RC,
MCM2–7 must be stably associated with the origin, and this stability is achieved
fol-lowing ATP hydrolysis, first by Cdc6 and then by ORC
Geminin Provides Another Control Over Replication Initiation
Gemininis a protein that provides another level of regulatory control over DNA
replication Geminin inhibits DNA replication by preventing the incorporation of
MCM complexes into the pre-RC Geminin is active during S, G2, and M phases, but
its destruction during mitosis permits replication initiation in G1 Geminin binds to
Cdt1, preventing it from recruiting MCM proteins to the pre-RC Geminin exists as
a parallel coiled-coil homodimer It interacts with Cdt1 in two ways: It has an array
of glutamate residues on its surface that interact with positive charges on Cdt1, and
an adjacent region on geminin interacts independently with the N-terminal region
of Cdt1
Eukaryotic Cells Contain a Number of Different DNA Polymerases
At least 19 different DNA polymerases have been described in eukaryotic cells thus
far These various polymerases have been assigned Greek letters in the order of their
discovery (Table 28.4 lists the principal ones) Multiple DNA polymerases participate
in leading and lagging-strand synthesis, but three—
P P
DNA
G 1 phase
Sld3
Sld2
Cdc45
Cdc45
Cdc45
S-CDK
MCM
Cdc7-Dbf-4
Cdc6 Cdt1
Origin of replication
Dpb11
P
S phase
Dpb11
Sld2 Sld2
Dpb11
ORC
MCM
P
ORC
P
FIGURE 28.12The initiation of DNA replication in eukaryotic cells Binding of the pre-RC to origins of repli-cation is followed by loading of MCM hexameric heli-cases, phosphorylation reactions mediated by S-CDK and Cdc7-Dbf4, and the binding of the 11-3-2 complex The passage of the cell from G 1 to S is defined by these events DNA polymerases are recruited to the origins of replication, where they can access single-stranded regions of DNA and initiate DNA synthesis.
Trang 2874 Chapter 28 DNA Metabolism: Replication, Recombination, and Repair
den DNA polymerase ␣ has an associated primase subunit and functions in the
initi-ation of nuclear DNA repliciniti-ation Given a template, it not only synthesizes an RNA primer of about 10 nucleotides, but it then adds 20 to 30 deoxynucleotides to extend the chain in the 5→3 direction DNA polymerase ␦, a heterotetrameric enzyme, is
the principal DNA polymerase in eukaryotic DNA replication It interacts with PCNA
protein (PCNA stands for proliferating cell nuclear antigen) Through its association with PCNA, DNA polymerase carries out highly processive DNA synthesis PCNA is
the eukaryotic counterpart of the E coli 2-sliding clamp; it clamps DNA polymerase
to the DNA Like 2, PCNA encircles the double helix, but in contrast to the prokary-otic2-sliding clamp, PCNA is a homotrimer, not a homodimer (Figure 28.13) DNA
polymerase⑀ also plays a major role in DNA replication It has an acidic C-terminal
extension lacking in other DNA polymerases and evidence suggests that this domain
is a sensor for DNA damage checkpoint control, halting DNA replication until the
damage is repaired DNA polymerase ␥ is the DNA-replicating enzyme of
mitochon-dria; DNA polymerase  functions in DNA repair The more recently discovered
eu-karyotic DNA polymerases (including , , , , and Rev1) are novel in that they are
more error-prone, resulting in lower fidelity of DNA replication Nevertheless, they have the important ability to function in DNA replication and repair when damaged regions of DNA are encountered
Other proteins involved in eukaryotic DNA replication include replication protein A (RPA), an ssDNA-binding protein that is the eukaryotic counterpart
of SSB, and replication factor C (RFC) RFC loads the PCNA sliding clamp onto
replicating DNA, thus acting as the eukaryotic counterpart of the prokaryotic
-complex.
Telomeres are the structures at the ends of eukaryotic chromosomes Telomeres are short (5 to 8 bp), tandemly repeated, G -rich nucleotide sequences that form protective caps 1 to 12 kbp long on the chromosome ends (see Chapter 11)
Ver-Subunits Polymerase Localization and Function (mass in kD) Subunit Function
DNA polymerase
Nuclear; initiation of nuclear DNA replication
DNA polymerase
Nuclear; principal polymerase in leading and lagging strand synthesis;
highly processive
DNA polymerase
Nuclear; leading and lagging strand synthesis, sensor of DNA damage checkpoint control
DNA polymerase
Mitochondria; mitochondrial DNA replication
DNA polymerase
DNA repair
TABLE 28.4 Biochemical Properties of the Principal Human DNA Polymerases
180 Catalytic subunit
68 Protein–protein interactions
125 Catalytic subunit
66 Structural
50 Interaction with PCNA
12 Protein–protein interactions
261 Catalytic subunit
59 Multimerization
17 Protein–protein interactions
12 Protein–protein interactions
140 Catalytic subunit
55 Processivity
39
(b)
(a)
x
x
FIGURE 28.13 Structure of the human PCNA
homo-trimer (a) Ribbon representation of the PCNA trimer
(pdb id 1AXC) with an axial view of a B-form DNA
duplex in its center The molecular mass of each PCNA
monomer is 37 kD (b) Molecular surface of the PCNA
trimer-DNA complex.
Trang 3tebrate telomeres have a TTAGGG consensus sequence Telomeres are necessary
for chromosome maintenance and stability DNA polymerases cannot replicate the
extreme 5-ends of chromosomes because these enzymes require a template and a
primer and replicate only in the 5→3 direction Thus, lagging strand synthesis at
the 3-ends of chromosomes is primed by RNA primase to form Okazaki fragments,
but these RNA primers are subsequently removed, resulting in gaps in the progeny
5-terminal strands at each end of the chromosome after each round of replication
(“primer gap”; see Figure 28.14)
Telomerase is an RNA-dependent DNA polymerase Telomerase maintains
telomere length by restoring telomeres at the 3-ends of chromosomes The RNA
upon which telomerase activity depends is actually part of the enzyme’s structure
That is, telomerase is a ribonucleoprotein, and its RNA component contains a
9- to 30-nucleotide-long region that serves as a template for the synthesis of
telom-eric repeats at DNA ends The human telomerase RNA component is 450
nucleo-tides long; its template sequence is CUAACCCUAAC (base-pairs with TTAGGG)
Telomerase uses the 3-end of the DNA as a primer and adds successive TTAGGG
(b)
Telomerase
5
DNA polymerase
5
5 Primer gap
3
3
3
(a)
Primer gap
+
3
5
3
5
+
3
5
FIGURE 28.14 Telomere replication (a) In replication of the lagging strand, short RNA primers are added (pink)
and extended by DNA polymerase When the RNA primer at the 5 -end of each strand is removed, there is no
nucleotide sequence to read in the next round of DNA replication The result is a gap (primer gap) at the 5 -end
of each strand (only one end of a chromosome is shown in this figure) (b) Asterisks indicate sequences at the
3 -end that cannot be copied by conventional DNA replication Synthesis of telomeric DNA by telomerase
extends the 5 -ends of DNA strands, allowing the strands to be copied by normal DNA replication.
HUMAN BIOCHEMISTRY
Telomeres—A Timely End to Chromosomes?
Mammalian cells in culture undergo only 50 or so cell divisions
be-fore they die Somatic cells are known to lack telomerase activity,
and thus, they inevitably lose bits of their telomeres with each cell
division Telomerase activity is missing because the telomerase–
reverse transcriptase gene (the TRT gene) is switched off This fact
has led to a telomere theory of cell aging, which suggests that cells
senesce and die when their telomeres are gone In support of this
notion, a team of biologists headed by Calvin B Harley at Geron Corporation used recombinant DNA techniques to express the cat-alytic subunit of human telomerase in skin cells in culture and ob-served that such cells divide 40 times more after cells lacking this treatment have become senescent These results, although contro-versial, may have relevance to the aging process
Trang 4876 Chapter 28 DNA Metabolism: Replication, Recombination, and Repair
repeats to it, employing its RNA as template over and over again (Figure 28.14, see also the figure in the Chapter 11 Human Biochemistry box Telomeres and Tumors)
Many viruses have genomes composed of RNA, not DNA How then is the informa-tion in these RNA genomes replicated? In 1964, Howard Temin noted that in-hibitors of DNA synthesis prevented infection of cells in culture by RNA tumor viruses such as avian sarcoma virus On the basis of this observation, Temin
pro-posed that DNA is an intermediate in the replication of such viruses; that is, an RNA
tumor virus can use viral RNA as the template for DNA synthesis.
RNA viral chromosome⎯⎯→ DNA intermediate ⎯⎯→ RNA viral chromosome
In 1970, Temin and David Baltimore independently discovered a viral enzyme
ca-pable of mediating such a process, namely, an RNA-directed DNA polymerase or, as
it is usually called, reverse transcriptase All RNA tumor viruses contain such an
en-zyme within their virions (viral particles) RNA viruses that replicate their RNA
genomes via a DNA intermediate are classified as retroviruses.
Like other DNA and RNA polymerases, reverse transcriptase synthesizes polynu-cleotides in the 5→3 direction, and like all DNA polymerases, reverse transcriptase requires a primer Interestingly, the primer is a specific tRNA molecule captured by the virion from the host cell in which it was produced The 3-end of the tRNA is base-paired with the viral RNA template at the site where DNA synthesis initiates, and its free 3-OH accepts the initial deoxynucleotide once transcription commences Re-verse transcriptase then transcribes the RNA template into a complementary DNA (cDNA) strand to form a double-stranded DNA⬊RNA hybrid
The Enzymatic Activities of Reverse Transcriptases
Reverse transcriptases possess three enzymatic activities, all of which are essential to viral replication:
1 RNA-directed DNA polymerase activity, for which the enzyme is named (see Figure
12.10)
2 RNase H activity Recall that RNase H is a nuclease that specifically degrades RNA
chains in DNA⬊RNA hybrids (see Figure 12.10) The RNase H function of re-verse transcriptase is an exonuclease activity that degrades the template genomic RNA and also removes the priming tRNA after DNA synthesis is completed
3 DNA-directed DNA polymerase activity This activity replicates the ssDNA remaining
af-ter RNase H degradation of the viral genome, yielding a DNA duplex This DNA du-plex directs the remainder of the viral infection process or becomes integrated into
A DEEPER LOOK
RNA as Genetic Material
Whereas the genetic material of cells is dsDNA, virtually all plant
viruses, several bacteriophages, and many animal viruses have
genomes consisting of RNA In most cases, this RNA is single
stranded Viruses with single-stranded genomes use the single
strand as a template for synthesis of a complementary strand,
which can then serve as template in replicating the original strand
Retroviruses are an interesting group of eukaryotic viruses with
single-stranded RNA genomes that replicate through a dsDNA in-termediate Furthermore, the life cycle of retroviruses includes an obligatory step in which the dsDNA is inserted into the host cell genome in a transposition event Retroviruses are responsible for
many diseases, including tumors and other disorders HIV-1, the human immunodeficiency virus that causes AIDS, is a retrovirus
Trang 5the host chromosome, where it can lie dormant for many years as a provirus
Acti-vation of the provirus restores the infectious state
HIV reverse transcriptase is of great clinical interest because it is the enzyme for
replication of the AIDS virus DNA synthesis by HIV reverse transcriptase is blocked
by nucleotide analogs such as AZT and 3TC (Figure 28.15) HIV reverse
transcrip-tase incorporates these analogs into growing DNA chains in place of dTMP (in the
case of AZT) or dCMP (in the case of 3TC) Once incorporated, these analogs block
further chain elongation because they lack a 3-OH where the next incoming dNTP
can be added HIV reverse transcriptase is error-prone: It incorporates the wrong
base at a frequency of 1 per 2000 to 4000 nucleotides polymerized This high error
rate during replication of the HIV genome means that the virus is ever changing, a
feature that makes it difficult to devise an effective vaccine
by Genetic Recombination?
Genetic recombinationis the natural process by which genetic information is
re-arranged to form new associations For example, compared to their parents,
prog-eny may have new combinations of traits because of genetic recombination At the
molecular level, genetic recombination is the exchange (or incorporation) of one
DNA sequence with (or into) another When recombination involves reaction
be-tween very similar sequences (homologous sequences) of DNA, the process is called
homologous recombination.When very different nucleotide sequences recombine,
it’s nonhomologous recombination Transposition—the enzymatic insertion of a
transposon (a mobile segment of DNA, see pages 885–886) into a new location in the
genome—and nonhomologous recombination (incorporation of a DNA segment
whose sequence differs greatly from the DNA at the point of insertion) are two types
of recombination that play a significant evolutionary role Nonhomologous
recom-bination occurs at a low frequency in all cells and serves as a powerful genetic force
that reshapes the genomes of all organisms Homologous recombination involves
an exchange of DNA sequences between homologous chromosomes, resulting in
the arrangement of genes into new combinations Homologous recombination is
generally used to fix the DNA so that information is not lost For example, large
le-sions in DNA are repaired via recombination of the damaged chromosome with a
homologous chromosome
The process underlying homologous recombination is termed general
recombi-nationbecause the enzymatic machinery that mediates the exchange can use
essen-tially any pair of homologous DNA sequences as substrates Homologous
recombi-nation occurs in all organisms and is particularly prevalent during the production of
gametes (meiosis) in diploid organisms In higher animals—that is, those with
im-mune systems—recombination also occurs in the DNA of somatic cells responsible
for expressing proteins of the immune response, such as the immunoglobulins This
somatic recombinationrearranges the immunoglobulin genes, dramatically
increas-ing the potential diversity of immunoglobulins available from a fixed amount of
ge-netic information Homologous recombination can also occur in bacteria Indeed,
even viral chromosomes undergo recombination For example, if two mutant viral
particles simultaneously infect a host cell, a recombination event between the two
viral genomes can lead to the formation of a virus chromosome that is wild-type
General Recombination Requires Breakage and Reunion
of DNA Strands
Recombination occurs by the breakage and reunion of DNA strands so that a
phys-ical exchange of parts takes place Matthew Meselson and J J Weigle demonstrated
this in 1961 by coinfecting E coli with two genetically distinct bacteriophage
CH3 O
O
O
N N H
O
H H H
HOCH2
N
N O
S
H H H H
HOCH2
N N
+ –
NH2 N
(a)
(b)
FIGURE 28.15 The structures of AZT (3 -azido-2,3-dideoxythymidine) and 3TC (2 ,3-dideoxy-3-thiacytidine) These nucleosides are phosphorylated
in vitro to form deoxynucleoside-5’-triphosphate sub-strate analogs for HIV reverse transcriptase.
Trang 6878 Chapter 28 DNA Metabolism: Replication, Recombination, and Repair
strains, one of which had been density-labeled by growth in 13C- and 15N-containing media (Figure 28.16) The phage progeny were recovered and separated by CsCl density gradient centrifugation Phage particles that displayed recombinant geno-types were distributed throughout the gradient while parental (nonrecombinant) genotypes were found within discrete “heavy” and “light” bands in the density gra-dient The results showed that recombinant phage contained DNA derived in vary-ing proportions from both parents The obvious explanation is that these recombi-nant DNAs arose via the breakage and rejoining of DNA molecules
A second important observation made during this type of experiment was that some of the plaques formed by the phage progeny contained phage of two differ-ent genotypes, even though each plaque was caused by a single phage infecting one bacterium Therefore, some infecting phage chromosomes must have contained a
region of heteroduplex DNA, duplex DNA in which a part of each strand is
con-tributed by a different parent (Figure 28.17)
Homologous Recombination Proceeds According
to the Holliday Model
In 1964, Robin Holliday proposed a model for homologous recombination that has proved to be correct in its essential features (Figure 28.18) The two homologous DNA duplexes are first juxtaposed so that their sequences are aligned This process
of chromosome pairing is called synapsis (Figure 28.18a) Holliday suggested that
recombination begins with the introduction of single-stranded nicks in the DNA at homologous sites on the two paired chromosomes (Figure 28.18b) The two du-plexes partially unwind, and the free, single-stranded end of one duplex begins to base-pair with its nearly complementary, single-stranded region along the intact
strand in the other duplex, and vice versa (Figure 28.18c) This strand invasion is
followed by ligation of the free ends from different duplexes to create a
cross-Infect bacteria with a mixture of heavy and light phage
Recombination occurs
Phage assembly and cell lysis
Heavy phage ABC Light phage abc
Phage progeny are centrifuged on CsCl gradient
Light phage abc
Recombinant phage Abc, aBc, abC, etc.
Heavy phage ABC
䊴 ANIMATED FIGURE 28.16 Meselson and Weigle’s experiment Density-labeled,“heavy” phage, symbolized as ABC phage, was used to coinfect bacteria along with “light” phage, the abc phage The progeny from the infection were collected and subjected to CsCl density gradient centrifugation Parental-type ABC and abc phage were well separated in the gradient, but recombinant phage (ABc, Abc, aBc, aBC, and so on) were distributed diffusely between the two parental bands because they contained
chromosomes constituted from fragments of both “heavy” and “light” DNA See this figure animated at
www.cengage.com/login.
+ –
Heteroduplex DNA (+ and – strands)
Semiconservative DNA replication
+ –
Progeny phage of 2 different genotypes
+ –
FIGURE 28.17 The generation of progeny bacteriophage of two different genotypes from a single phage parti-cle carrying a heteroduplex DNA region within its chromosome The heteroduplex DNA is composed of one strand that is genotypically XYZ (the strand), and the other strand that is genotypically XyZ (the strand) That is, the genotype of the two parental strands for gene Y is different (one is Y, the other y).
Trang 7stranded intermediate known as a Holliday junction (Figure 28.18d) The
cross-stranded junction can now migrate in either direction (branch migration) by
un-winding and reun-winding of the two duplexes (Figure 28.18e) Branch migration
re-sults in strand exchange; heteroduplex regions of varying length are possible In
order for the joint molecule formed by strand exchange to be resolved into two
DNA duplex molecules, another pair of nicks must be introduced Resolution can
+ – +
Nicking
Strand invasion
Ligation
Branch migration
Arms conceptually bent
“up” and “down”
+–
+ – – +
+ – – +
(–) strands cleaved at junction, strands resolved, religated
(+) strands cleaved at junction, strands resolved, religated
Patch recombinant heteroduplex Splice recombinant heteroduplex
(a)
(b)
(c)
(d)
(e)
(f)
N
S
ACTIVE FIGURE 28.18 The Holliday model for homologous recombination The signs and signs label strands of like polarity For exam-ple, assume that the two strands running 5 →3 as read left to right are labeled ; and the two strands running 3 →5 as read left to right are labeled Only strands of like polarity exchange DNA during recombination (See text for detailed description.)
Test yourself on the concepts in this figure at www.cengage.com/login.
Trang 8880 Chapter 28 DNA Metabolism: Replication, Recombination, and Repair
be represented best if the duplexes are drawn with the chromosome arms bent “up”
or “down” to give a planar representation (Figure 28.18f) Nicks then take place, ei-ther at E and W, that is, in the strands that were originally nicked (see Figure 28.18b), or at N and S, that is, in the strands (the strands not previously nicked) Duplex resolution is most easily kept straight by remembering that strands are complementary to strands and any resultant duplex must have one of each Nicks made in the strands originally nicked lead to DNA duplexes in which one strand of each remains intact Although these duplexes contain heteroduplex regions, they are not recombinant for the markers (AZ, az) that flank the heteroduplex region;
such heteroduplexes are called patch recombinants (Figure 28.18g) Nicks
intro-duced into the two strands not previously nicked yield DNA molecules that are both heteroduplex and recombinant for the markers A/a and Z/z; these heteroduplexes
are termed splice recombinants (Figure 28.18h) Although this Holliday model
ex-plains the outcome of recombination, it provides no mechanistic explanation for the strand exchange reactions and other molecular details of the process
The Enzymes of General Recombination Include RecA, RecBCD, RuvA, RuvB, and RuvC
To illustrate recombination mechanisms, we focus on general recombination as it
occurs in E coli The principal players in the process are the RecBCD enzyme
com-plex, which initiates recombination; the RecA protein, which binds single-stranded
DNA, forming a nucleoprotein filament capable of strand invasion and
homolo-gous pairing; and the RuvA, RuvB, and RuvC proteins, which drive branch
migra-tion and process the Holliday juncmigra-tion into recombinant products Eukaryotic homologs of these prokaryotic recombination proteins have been identified, indi-cating that the fundamental process of general recombination is conserved across all organisms
The RecBCD Enzyme Complex Unwinds dsDNA and Cleaves Its Single Strands
The RecBCD complex is composed of the proteins RecB (140 kD; 1180 amino acids), RecC (130 kD; 1122 amino acids), and RecD (67 kD; 608 amino acids) This
multi-functional enzyme complex has both helicase and nuclease activity and initiates re-combination by attaching to the end of a DNA duplex (or at a double-stranded break
in the DNA) and using its ATP-dependent helicase function to unwind the dsDNA (Figure 28.19a) RecB and RecD are helicases powered by ATP hydrolysis; each con-sumes an ATP per base pair of DNA traversed The RecD motor is faster than the RecB motor and leads the way The greater speed of RecD causes the DNA to loop out between RecD and RecB The RecB subunit contains the nuclease domain As RecBCD progresses along unwinding the duplex, the RecB nuclease activity cleaves both of the newly formed single strands (although the strand that provided the 3-end
at the RecBCD entry site is cut more frequently than the 5-terminal strand [Figure 28.19b]) SSB (and some RecA protein) readily binds to the emerging single strands
Sooner or later, RecBCD encounters a particular nucleotide sequence, a so-called Chi
re-combinational “hot spots”; 1009 sites have been identified in the E coli genome
(on average, about one every 4.5 kb of DNA) When a sequence is encountered by
a RecBCD complex approaching its 3-side (the G-3-side), RecBCD cleaves the
-bearing DNA strand four to six bases to the 3 side of (Figure 28.19c) RecBCD
flips so that the RecB motor leads the way, and the RecBCD complex no longer ex-presses nuclease activity against the 3-terminal strand, but nuclease activity against the 5-terminal strand increases (Figure 28.19d)
Resuming its helicase function, RecBCD unwinds the dsDNA, and collectively these processes generate an ssDNA tail bearing a site at its 3-terminal end
This ssDNA may reach several kilobases in length RecA protein now binds to the 3-terminal strand to form a nucleoprotein filament (Figure 28.19e) This filament
Trang 9is active in pairing and strand invasion with a homologous region in another dsDNA
molecule
The RecA Protein Can Bind ssDNA and Then Interact with Duplex DNA
The RecA protein, or recombinase, is a multifunctional protein that acts in general
recombination (Figure 28.20a) RecA mediates the ATP-dependent DNA strand
exchange reactionleading to formation of a Holliday junction (Figure 28.18b–f) In
the presence of ATP and ssDNA, RecA forms a right-handed helical filament having
(a)
(b)
(c)
(d)
(e)
3
3
3
3
5
5
5
5
3
5
3
5
3
5
3 5
3
5
3
5
χ
3
5
3
5
χ
χ
χ
χ
RecBCD
SSB RecA
RecA
Binding of RecBCD to DNA end; unwinding
Nonspecific cleavage (3 more than 5);
SSB, RecA binding
χ encounter and cleavage 3 to χ
Displacement of SSB;
preferential binding of RecA;
enhanced 5 -terminal cleavage
Pairing with homologous DNA duplex and strand invasion
χ
FIGURE 28.19 Model of RecBCD-dependent initiation of
recombination (a) RecBCD binds to a duplex DNA end,
and its helicase activity begins to unwind the DNA dou-ble helix.“Rabbit ears” of ssDNA loop out from RecBCD because the rate of DNA unwinding exceeds the rate of
ssDNA release by RecBCD (b) As it unwinds the DNA,
SSB (and some RecA) bind to the single-stranded re-gions; the RecBCD endonuclease activity randomly cleaves the ssDNA, showing a greater tendency to cut the 3 -terminal strand rather than the 5-terminal strand.
(c) When RecBCD encounters a properly oriented site,
the 3 -terminal strand is cleaved just below the 3-end
of (d) RecBCD now directs the binding of RecA to the
3 -terminal strand, as RecBCD endonuclease activity now acts more often on the 5-terminal strand (e) A
nucleo-protein filament consisting of RecA-coated 3 -strand ssDNA is formed This nucleoprotein filament is capable
of homologous pairing with a dsDNA and strand inva-sion (Adapted from Figure 2 in Eggleston, A K., and West, S C.,
1996 Exchanging partners: recombination in E coli.Trends in
Genetics 12:20–25; and Figure 3 in Eggleston, A K., and West, S C.,
1997 Recombination initiation: Easy as A, B, C, D …. ? Current
Biology 7:R745–R749.)
Trang 10882 Chapter 28 DNA Metabolism: Replication, Recombination, and Repair
six monomers per turn, with each monomer spanning about three nucleotides of DNA The RecA nucleoprotein filament serves as a scaffold upon which the events of recombination take place This filament has a deep spiral groove large enough to en-compass three strands of DNA Although RecA-bound ssDNA is relatively stretched and underwound, with about 18.5 nucleotides per turn, its local conformation re-sembles B-DNA (Figure 28.20b) Thus, when dsDNA adds to the complex, the ssDNA
is poised to search for the sequence homology that leads to strand exchange Procession of strand separation of dsDNA and the re-pairing into hybrid strands
along the DNA duplex initiates branch migration (Figure 28.21b) Branch migration
drives the displacement of the homologous DNA strand from the DNA duplex and
its replacement with the ssDNA strand, a process known as single-strand assimilation
(b)
C-term
N-term
(a)
L1
FIGURE 28.20 (a) RecA, a 352-residue, 38-kD protein,
with dATP in the ATP-binding site (pdb id 2ODN).
(b) Structure of a fused RecA hexamer with bound ssDNA
(as poly(dT) 18 , magenta) Each of the six RecA units is a
dif-ferent color and each has a bound ADP molecule (marine
blue).The adenine nucleotide-binding sites lie at the
inter-faces between RecA units (pdb id 3CMU).
5
3
5
3
3
5
3
5
3
5
5
3
5
5
5
3
5
3
5
3
3
5
(a)
RecA
Homologous DNA duplex
(b)
(c)
SSB
FIGURE 28.21 Model for homologous recombination as promoted by RecA enzyme (a) RecA protein (and SSB) aid
strand invasion of the 3-ssDNA into a homologous DNA duplex, (b) forming a D-loop.(c) The D-loop strand that
has been displaced by strand invasion pairs with its complementary strand in the original duplex to form a Holli-day junction as strand invasion continues.