Báo cáo khoa học: Ribonuclease H: molecular diversities, substrate binding domains, and catalytic mechanism of the prokaryotic enzymes ppt

Ribonuclease H: molecular diversities, substrate binding domains, and catalytic mechanism of the prokaryotic enzymes Takashi Tadokoro and Shigenori Kanaya Department of Material and Life

Ribonuclease H: molecular diversities, substrate binding domains, and catalytic mechanism of the prokaryotic

enzymes

Takashi Tadokoro and Shigenori Kanaya

Department of Material and Life Science, Osaka University, Japan

Identification of bacterial RNase HIII

with a TBP-like substrate-binding

domain

RNase H is defined as an enzyme that specifically

hydrolyzes the phosphodiester bonds of RNA

hybrid-ized to DNA at the P–O3¢ bond Prokaryotic

RNases H, which are involved in DNA replication,

repair and transcription [1,2] have been classified into

RNases HI, HII and HIII based on differences in

their amino acid sequences [3,4] (Fig 1) RNase HI

represents type 1 RNase H, and RNases HII and

HIII represent type 2 RNase H The genes encoding

RNase HI [5] and RNase HII [6] were first cloned

from the Escherichia coli genome in 1983 and 1990,

respectively The gene encoding RNase HIII was first cloned from the Bacillus subtilis genome in 1999 [3] Two different terminologies, RNases HII and HIII, are given to type 2 RNases H because certain bacte-rial genomes, such as the B subtilis, Streptococ-cus pneumoniae and Aquifex aeolicus genomes, contain two genes encoding type 2 RNases H The protein that shows higher amino acid sequence similarity

to E coli RNase HII is designated as RNase HII, whereas the other is designated as RNase HIII Unlike RNase HII, which is widely present in various organisms including bacteria and archaea, RNase HIII is present only in a limited number of bacteria [3,4]

Keywords

catalytic mechanism; crystal structure;

evolution; genome; hybrid binding domain;

molecular diversity; prokaryote; RNase H;

RNA ⁄ DNA hybrid; substrate-binding domain

Correspondence

S Kanaya, Department of Material and Life

Science, Graduate School of Engineering,

Osaka University, 2-1, Yamadaoka, Suita,

Osaka 565-0871, Japan

Fax ⁄ Tel: +81 6 6879 7938

E-mail: kanaya@mls.eng.osaka-u.ac.jp

(Received 18 October 2008, revised 18

December 2008, accepted 12 January 2009)

doi:10.1111/j.1742-4658.2009.06907.x

The prokaryotic genomes, for which complete nucleotide sequences are available, always contain at least one RNase H gene, indicating that RNase H is ubiquitous in all prokaryotic cells Coupled with its unique substrate specificity, the enzyme has been expected to play crucial roles in the biochemical processes associated with DNA replication, gene expression and DNA repair The physiological role of prokaryotic RNases H, espe-cially of type 1 RNases H, has been extensively studied using Escherichia coli strains that are defective in RNase HI activity or overproduce RNase HI However, it is not fully understood yet By contrast, significant progress has been made in this decade in identifying novel RNases H with respect to their biochemical properties and structures, and elucidating catalytic mechanism and substrate recognition mechanism of RNase H

We review the results of these studies

Abbreviations

Afu, Archaeoglobus fulgidus; Bha, Bacillus halodurans; Bst, Bacillus stearothermophilus; Bsu, Bacillus subtilis; Eco, Escherichia coli; Halo, Halobacterium sp NRC-1; HBD, hybrid binding domain; HIV-1, human immunodeficiency virus type 1; Mja, Methanococcus jannaschii; MMLV, Moloney murine leukemia virus; Pae, Pyrobaculum aerophilum; RNase H, ribonuclease H; Son, Shewanella oneidensis; Sto, Sulfolobus tokodaii; TBP, TATA-box binding proteins; Tko, Thermococcus kodakaraensis; Tma, Thermotoga maritima; Tth,

Thermus thermophilus.

The enzymatic properties of RNase HIII have been

well characterized using B subtilis RNase HIII

(Bsu-RNase HIII) [3] (Bsu-RNase HIII is evolutionarily more

distantly related to RNase HI than to RNase HII For

example, Bsu-RNase HIII shows the poor amino

acid sequence identity to E coli RNase HI

(Eco-RNase HI), whereas it shows the amino acid sequence

identities of 20–21% to E coli RNase HII

(Eco-RNase HII) and B subtilis (Eco-RNase HII (Bsu-(Eco-RNase

HII) Nevertheless, Bsu-RNases HIII is more closely

related to Eco-RNase HI than to Eco-RNase HII or

Bsu-RNase HII in the enzymatic properties, such as

metal ion specificities, specific activities and cleavage

site specificities These results may suggest that

Bsu-RNase HIII functions as a substitute for

Eco-RNase HI However, it has been reported for

Chlamydophila pneumoniae that the RNase HII⁄ HIII

combination is not a simple substitution of Eco-RNase

HI⁄ HII [7,8]

RNases HIII are characterized by the presence of a long N-terminal extension compared with other type 2 RNases H (Fig 1) The amino acid sequences of these extensions show a significant similarity to those of TATA-box binding proteins (TBPs) The first crystal structure of RNase HIII was determined using RNase HIII from the moderate thermophile B stearo-thermophilus (Bst-RNase HIII) [9] Bst-RNase HIII shows the amino acid sequence identity of 47.1% to Bsu-RNase HIII and has a similar N-terminal exten-sion Its enzymatic properties are similar to those of Bsu-RNase HIII, except that it is more stable [10] According to the crystal structure of Bst-RNase HIII, the N-terminal extension assumes a TBP-like structure and is present as an independent domain (Fig 2) The

Fig 1 Schematic representation of the

pri-mary structures of prokaryotic RNases H.

The primary structures of the representative

members of type 1 (A) and type 2 (B)

RNases H, which have been reported to be

enzymatically active, are shown Solid box

represents the RNase H domain and gray

box represents TBP-like domain,

hybrid-bind-ing domain (HBD) or acid phosphatase

domain The positions of the four acidic

active site residues and the histidine

resi-due, which is well conserved in type 1

RNases H, are shown The numbers

repre-sent the positions of the amino acid

residues relative to the initiator methionine

for each protein.

structure of the C-terminal RNase H domain is highly

similar to those of archaeal RNases HII [11–13]

(Fig 2) The active site motif of RNase HIII (DEDE)

is similar, but is not identical to that of RNase HI or

HII (DEDD) [4] (Fig 1) Nevertheless, the steric

configurations of these four acidic active-site residues

are very similar to those of RNases HI and HII

Bio-chemical characterization of the Bst-RNase HIII

deriv-atives with N- and⁄ or C-terminal truncations indicates

that the N-terminal domain with a TBP-like structure

and C-terminal helix are involved in substrate binding,

but the former contributes to substrate binding more

greatly than the latter [9] TBP binds to the target

DNA at the flat surface of the molecule [14–16] The

N-terminal domain of RNase HIII probably uses the

same surface for substrate binding as TBP to bind

DNA

Identification of archaeal type 1 RNases H

Most of the archaeal genomes, for which the complete genome sequences are available, only contain the type 2 RNase H (RNase HII) genes The exceptions are the Halobacterium sp NRC-1, Sulfolobus tokodaii and Pyrobaculum aerophilum genomes, which contain the type 1 RNase H (RNase HI) genes in addition to the RNase HII genes The first archaeal RNase HI protein, which was shown to be active both in vivo and

in vitro, is RNase HI from Halobacterium sp NRC-1 (Halo-RNase HI) [17] Later, RNases HI from

S tokodaii (Sto-RNase HI) and P aerophilum (Pae-RNase HI) were also shown to be enzymatically active [18] Database searches using the Sto-RNase HI sequence indicate that not only the archaeal genomes,

Fig 1 (Continued).

but also the bacterial and eukaryotic genomes contain

the genes encoding a Sto-RNase HI homologue Of

them, the Sto-RNase HI homologue from B subtilis

(YpdQ) is inactive [3], whereas those from

Strepto-myces coelicolor(SCO7284 [18] and SCO2299 [19]) and

Corynebacterium glutamicum (Cg12236) [20] are active

Phylogenetic analyses of the type 1 RNase H

sequences show that the Sto-RNase HI homologues

form an independent domain, which is different from

those of bacterial, eukaryotic and retroviral type 1

RNases H [18] Characteristics common to the amino

acid sequences of these Sto-RNase HI homologues are

the lack of a basic protrusion, which has been shown

to be important for substrate binding in

Eco-RNase HI [21,22], and the lack of the histidine residue, which is well conserved in other type 1 RNases H and has been shown to be important for catalytic function

of Eco-RNase HI [23] (Fig 1)

Sto-RNase HI consists of 149 amino acid residues and has neither an N-terminal nor C-terminal extension compared with Eco-RNase HI (Fig 1) By contrast, Halo-RNase HI consists of 199 residues and has an N-terminal 65-residue extension (Fig 1) This extension does not show significant amino acid sequence similar-ity to an N-terminal TBP-like domain of RNases HIII

or hybrid-binding domain (HBD) of eukaryotic RNases H1 Sto-RNase HI and Halo-RNase HI exhibit unique enzymatic properties They cleave the substrate

at the RNA–DNA junction [17,18] Interestingly, Sto-RNase HI exhibits Sto-RNase H* activity, which degrades the RNA strand of the RNA⁄ RNA duplex [18] It has been reported that retroviral RNases H cleave the RNA–DNA junction [24] and exhibit RNase H* activ-ity [25] Thus, archaeal RNases HI are more closely related to retroviral RNases H than bacterial and eukaryotic type 1 RNases H in enzymatic properties The crystal structure of Sto-RNase HI was deter-mined as the first archaeal type 1 RNase H structure [26] (Fig 2) Despite the low amino acid sequence identity between Sto-RNase HI and other type 1 RNases H, Sto-RNase HI shows high structural simi-larity to these RNases H, including Eco-RNase HI [27,28], human immunodeficiency virus type 1 (HIV-1) RNase H [29,30] and Bacillus halodurans RNase HI (Bha-RNase HI) [31] The steric configurations of the four acidic active-site residues are well conserved in the Sto-RNase HI structure (Fig 3) Like other Sto-RNase HI homologues, Sto-RNase HI lacks a his-tidine residue, which is well conserved in various type 1 RNases H However, Arg118 of Sto-RNase HI

is located in the same position as His124 of Eco-RNase HI, His539 of HIV-1 RNase H and Glu188 of Bha-RNase H Mutation of this residue to Ala considerably reduces both the RNase H and RNase H* activities without seriously affecting sub-strate binding, suggesting that Arg118 is involved in catalytic function [26] This residue may promote prod-uct release by perturbing the coordination of the metal ion A, as proposed for Glu188 of Bha-RNase H [31] The catalytic mechanism of RNase H is described in more detail below

As described below under ‘Substrate recognition mechanism’, RNase H has two grooves in which the RNA and DNA backbones of RNA⁄ DNA hybrids bind A phosphate-binding pocket and DNA-binding channel located in the DNA-binding groove are respon-sible for the specificity of RNase H for the RNA⁄ DNA

Fig 2 Crystal structures of prokaryotic RNases H Ribbon

dia-grams of RNases HI from S tokodaii (Sto-RNase HI) (PDB code

2EHG) and E coli (Eco-RNase HI) (PDB code 2RN2), RNases HII

from T maritima (Tma-RNase HII) (PDB code 2ETJ) and T

kodaka-raensis (Tko-RNase HII) (PDB code 1IO2), and B

stearothermophi-lus RNase HIII (Bst-RNase HIII) (PDB code 2D0A) N and C

represent the N- and C-termini Four acidic active site residues are

shown as ball-and-stick models The basic protrusion of

Eco-RNa-se HI, and TBP-like domain and C-terminal helix of Bst-RNaEco-RNa-se HIII

are indicated.

hybrid However, the amino acid residues forming the

phosphate-binding pocket are not well conserved in the

Sto-RNase HI structure In addition, the DNA-binding

channel, which is formed in the basic protrusion, is not

present in the RNase HI structure because

Sto-RNase HI lacks a basic protrusion By contrast, the

RNA-binding groove is well conserved in the

Sto-RNase HI structure The weak conservation of the

DNA-binding groove may account for the ability of

Sto-RNase HI to cleave double-stranded (ds)RNA

Weaker conservation of the DNA-binding groove

com-pared with that of the RNA-binding groove is also

observed in HIV-1 and Moloney murine leukemia virus

(MMLV) RNases H

It is noted that the structure of Sto-RNase HI is

highly similar to that of the RNase H-like domain of

the PIWI domain of Argonaute from A aiolicus [32]

Unlike other Argonaute proteins, A aeolicus

Argona-ute exhibits both RNA-guided and DNA-guided

RNase activities, using the RNase H-like domain of

the PIWI domain [32]

Identification of bacterial RNase HI

with an N-terminal hybrid binding

domain

HBD-RNase HI from the psychrotrophic bacterium

Shewanella sp SIB1 is the first bacterial type 1

RNase H protein containing an N-terminal HBD to

be characterized biochemically [33] Bha-RNase HI

also contains HBD at the N-terminus [31] HBD,

which has previously been designated as a dsRNA

binding domain, consists of  50 amino acid residues

and is commonly present at the N-termini of

eukary-otic type 1 RNases H [34] This domain is renamed

as HBD, because HBD of human RNase H1 binds

more strongly to RNA⁄ DNA hybrids than to

dsRNA [35] The binding mechanism of HBD and

its role in enzymatic activity is discussed in more detail below, based on the co-crystal structure of HBD of human RNase H1 with the RNA⁄ DNA hybrid [35]

HBD-RNase HI from Shewanella sp SIB1, which has previously been designated as RBD-RNase HI [33], consists of 262 amino acid residues (Fig 1) The Shewanella sp SIB1 genome contains RNase HI [36] and RNase HII [37] genes as well All three RNase H proteins are enzymatically active Thus, this genome contains three active RNase H genes (two type 1 and one type 2) In addition to SIB1 genome, the S frigid-imarina, S denitrificans and Photobacterium profundum genomes contain three genes encoding RNase HI, HBD-RNase HI and RNase HII Of these, two type 1 RNases H, HBD-RNase HI (RNase H domain) always shows lower sequence identity to Eco-RNase HI For example, SIB1 HBD-RNase HI and SIB1 RNase HI show amino acid sequence identities of 17 and 63% to Eco-RNase HI, respectively It has been reported that the S coelicolor genome also contains three active RNase H genes encoding two type 1 and one type 2 RNases H [19] However, none of them contain HBD

at the N-termini Interestingly, one of these type 1 RNases H (SCO2299) is a bifunctional enzyme consist-ing of an N-terminal RNase H domain and a C-termi-nal acid phosphatase domain [19] (Fig 1)

Catalytic mechanism of RNase H

The crystal structures of eight type 1 and five type 2 RNases H have so far been determined They are Eco-RNase HI [27,28], Thermus thermophilus RNase HI (Tth-RNase HI) [38], Bha-RNase HI [31],

Shewanel-la oneidensis RNase HI (Son-RNase HI) [39], Sto-RNase HI [26], HIV-1 RNase H [29,30], MMLV RNase H [40,41] and human RNase H1 [42] for type 1 RNases H, and Methanococcus jannaschii RNase HII

Fig 3 Stereoview of the active site struc-tures of RNases H The side chains of the active site residues of Sto-RNase HI (salmon) and HIV-1 RNase H (green) are superimposed onto those in the co-crystal structure of Bha-RNase H with the sub-strate and metal ions (yellow) The positions

of the RNA strand of the substrate with the scissile phosphate group between R( )1) and R(0) and two metal ions A and B are also shown.

(Mja-RNase HII) [11], Thermococcus kodakaraensis

RNase HII (Tko-RNase HII) [12], Archaeoglobus

fulgi-dusRNase HII (Afu-RNase HII) [13], Bst-RNase HIII

[9] and Thermotoga maritima RNase HII (TmaRNase

-HII) (PDB code 2ETJ) for type 2 RNases H The

struc-tures of the representative members of these RNases H

are shown in Fig 2 These structures share a main chain

fold, termed the RNase H-fold, and steric

configura-tions of the four acidic active-site residues, suggesting

that their catalytic mechanisms are basically identical

It has long been controversial whether the enzyme

requires one or two metal ions for activity, because the

number of the metal-binding sites found in the crystal

structures of the enzyme–metal ion complex formed in

the absence of the substrate varies for different

enzymes and different metal ions For example, single

Mg2+[43] and Mn2+[44] ions bind to the active sites

of the wild-type and mutant proteins of

Eco-RNa-se HI, respectively, whereas two Mn2+ ions bind to

Eco-RNase HI [45] and HIV-1 RNase H [29]

How-ever, the co-crystal structure of Bha-RNase HI with

RNA⁄ DNA substrate and Mg2+ [31] shows that two

Mg2+ ions bind to the active site of the enzyme

(Fig 3) Both metal ions are coordinated by the acidic

active site residues, scissile phosphate group of the

substrate and water molecules The distance between

these two metal ions increases when the substrate is

cleaved [46] Based on these results, two-metal-ion

catalysis mechanism has been proposed for RNase H

[31,46,47] According to this mechanism, metal ion A

is required for substrate-assisted nucleophile formation

and product release, and metal ion B is required to

destabilize the enzyme–substrate complex and thereby

promote the phosphoryl transfer reaction (Fig 4)

However, it remains controversial whether an active

site carboxyl group directly participates in catalysis as

a general base [48]

In addition to the four acidic active site residues, the

histidine residue is well conserved in the type 1

RNase H sequences (His124 for Eco-RNase HI and

His539 for HIV-1 RNase H) (Fig 1) This residue is

located in the flexible loop near the active site and is

involved in the catalytic function, but in an auxiliary

manner [23] This residue is conserved as His264 in

human RNase HI, but is replaced by Glu188 in

Bha-RNase HI Based on the co-crystal structures of human

RNase H1 and Bha-RNase HI, in which this residue is

conserved as His264 and replaced by Glu188,

respec-tively, with the substrate and Mg2+, this residue has

been proposed to promotes the product release by

per-turbing the coordination of the metal ion A [31,42]

It is noted that a folding motif of RNases H, termed

RNase H-fold, has been found in other proteins with

nuclease or polynucleotidyl transferase activities, such

as integrase [49], DNA transposase [50], RuvC Holli-day junction resolvase [51], and a PIWI domain of Argonaute proteins [32,52,53], which is essential for RNA-induced silencing complex-mediated mRNA cleavage Because three or four acidic amino acid resi-dues also form the active sites of these enzymes, these enzymes may also share a catalytic mechanism with RNase H

Substrate recognition mechanism of RNase H

The crystal structure of human RNase H1 in complex with the substrate and Mg2+highly resembles to that of Eco-RNase HI free from the substrate [42], indicating that the structure of human RNase HI is not seriously changed upon substrate binding According to the co-crystal structure of human RNase HI with the sub-strate and Mg2+, the RNA⁄ DNA hybrid binds to the protein, such that the RNA backbone fits in one groove containing the active site and the DNA backbone fits in the other groove (Fig 5) These two grooves are sepa-rated by a ridge, which is composed of highly conserved Asn151, Asn182 and Gln183 (Asn16, Asn44 and Asn45

Fig 4 Schematic representation of the two-metal-ion catalysis mechanism proposed for RNase H The side chains of the first aspartate, second glutamate, third aspartate and fourth aspartate residues of the DEDD motif, which form the active site (metal-bind-ing site) of RNase H, are shown They are Asp10, Glu48, Asp70 and Asp134 for Eco-RNase HI, Asp71, Glu109, Asp132, and Asp192 for Bha-RNase HI, Asp145, Glu186, Asp210 and Asp274 for human RNase H1, and Asp443, Glu478, Asp498 and Asp549 for HIV-1 RNase H The fourth aspartate residue is replaced by the glutamate residue for RNase HIII The attacking hydroxyl ion that

is coordinated by metal ion A is highlighted in boldface type.

for Eco-RNase HI), and interacts with the minor groove

of the RNA⁄ DNA hybrid

At the RNA-binding groove, the 2¢-OH groups of

four consecutive ribonucleotides, two on each side

of the scissile phosphate group, contact the side chain

of Glu186 (Glu48 for Eco-RNase HI) and the

back-bone atoms of Cys148, Ser150, Asn151 and Met212

(Cys13, Gly15, Asn16 and Gln72 for Eco-RNase HI)

Similar contacts are observed between Bha-RNase HI

and the RNA strand of the substrate, indicating that

the mechanism for RNA strand recognition is

con-served among various type 1 RNases H The

DNA-binding groove contains two DNA-DNA-binding sites The

major site is a phosphate-binding pocket, which is

formed by Arg179, Thr181 and Asn240 (Arg41, Thr43

and Asn100 for Eco-RNase HI) and is conserved in

the Bha-RNase HI structure This site is responsible

for anchoring the B-form DNA The second site is a

channel formed by Trp221, Trp225 and Ser233 (Trp81,

Trp85 and Ala93 for Eco-RNase HI) in the basic

protrusion This site is absent in the Bha-RNase HI,

because Bha-RNase HI lacks a basic protrusion The

RNA strand cannot fit in this groove, because a 2¢-OH

group of RNA clashes with the indole ring of Trp221

Therefore, these two DNA binding sites probably

con-tribute to the specificity for an RNA⁄ DNA hybrid

High resemblance of the structures of human

RNase H1 and Eco-RNase HI strongly suggests that

these proteins recognize the substrate in a similar mechanism (Fig 5) In fact, the mutational studies suggest that Cys13, Asn16, Gln72, Thr43 and Trp85 of Eco-RNase HI are important for substrate binding [54] Mutational studies also suggest that basic amino acid residues clustered in the basic protrusion are important for substrate binding [21] However, none of these basic residues makes direct contact with the sub-strate according to the co-crystal structure of human RNase H1 with the substrate It has been suggested that these basic residues facilitate initial nonspecific interactions with the substrate and promote the forma-tion of specific enzyme–substrate complexes [42]

It is noted that type 2 RNases H differ from type 1 RNases H in the location of a domain involved in sub-strate binding For example, Tko-RNase HII and Bst-RNase HIII have a substrate-binding domain at the C- and N-termini, respectively Both proteins lack

a basic protrusion Further mutational and structural studies will be required to understand the substrate recognition mechanism of these type 2 RNases H

Multiplicity of the RNase H genes in the single genomes

Single bacterial genomes often contain multiple RNase H genes For example, the E coli genome con-tains two genes encoding RNases HI and HII [6] The

B subtilis genome contains three genes encoding one type 1 RNase H with an N-terminal HBD-like domain (RNase HI) [55] and two type 2 RNases H (RNases HII and HIII) [3] By contrast, the archaeal genomes usually contain single RNase HII genes The exceptions are the genomes of S tokodaii [18] and Halobacterium sp NRC-1 [17] These genomes contain genes encoding RNases HI in addition to those encod-ing RNases HII The question arises whether the number and types of the RNase H genes contained in the single bacterial genomes are correlated with the evolutionary relationships of their source organisms determined based on the 16S rRNA sequences The answer is no For example, Shewanella sp SIB1 and

S oneidensisMR-1 are c-proteobacteria and evolution-arily closely related Nevertheless, the former genome contains three genes encoding RNase HI, HBD-RNase HI and HBD-RNase HII, whereas the latter contains only two genes encoding RNases HI and HII [33] Likewise, T maritima and A aeolicus are hypertherm-ophilic bacteria and evolutionarily closely related Nevertheless, the former genome contains the RNase HI and RNase HII genes, whereas the latter contains the RNases HII and HIII genes [4] These results suggest that RNase H genes have been

Fig 5 Crystal structure of the RNase H–substrate complex The

crystal structure of Eco-RNase HI (PDB code 2RN2) is

superim-posed onto that of the human RNase H1 C-domain complexed with

RNA ⁄ DNA hybrid (PDB code 2QK9) The structures of

Eco-RNa-se HI and human RNaEco-RNa-se H1 C domain are shown in green and

gold, respectively The active-site residues are shown as red

ball-and-stick models The basic protrusion and the phosphate binding

pocket and DNA-binding channel in the DNA-binding groove are

also indicated.

transferred horizontally among different organisms

during evolutionary processes

The physiological significance of the multiplicity of

RNases H in cells remains to be understood The

observation that RNase HII⁄ H2 cleaves dsDNA containing single ribonucleotide at the DNA–RNA junction (5¢ side of the ribonucleotide) [56,57] suggests that RNase HII⁄ H2 is involved in the excision of a

Table 1 Diversity of RNase H in different organisms a

HI ⁄ H1 without HBD HI ⁄ H1 with HBD HII ⁄ H2 HIII Bacteria

(Actinobacteria)

(Chlamydiae)

(Firmicutes)

(Proteobacteria)

(Others)

Archaea

Eukaryotes

Q09633 (rnh-1.1) Q23676 (rnh-1.2)

Q5ABY6 (RNH12) Q5ABY8 (RNH13) Retrovirus

Human immunodeficiency virus type 1 A U53871 f

a The accession numbers for RNases HI ⁄ H1 with and without HBD, RNases HII ⁄ H2, and RNases HIII are shown Only the representative members of organisms with fifferent combination of the RNase H genes are listed The genes A, A¢, B, and C represent those encoding RNases HI ⁄ H1 without HBD, RNases HI ⁄ H1 with HBD, RNases HII ⁄ H2, and RNases HIII, respectively The accession numbers for the pro-teins which do not exhibit RNase H activity or exhibits other activities are underlined b N-terminal domain of bi-functional enzyme c It has

an N-terminal extension with little sequence similarity to HBD and TBP.dIt has a C-terminal extension.eCatalytic subunit of a heterotrimer.

f C-terminal domain of reverse transcriptase.

single ribonucleotide misincorporated into DNA.

RNase HI⁄ H1 is not involved in this process because

it does not cleave this substrate However, all RNases H

endonucleolytically cleave RNA⁄ DNA hybrids in a

nonspecific manner The T thermophilus RNase HII

orthologue does not exhibit RNase H activity but

cleaves the RNA–DNA junction, and is therefore

designated as junction ribonuclease [58]

Functional similarities between type 1 and type 2

RNases H may suggest that a physiological

signifi-cance of the multiplicity of the RNase H genes in a

single genome is the protection of cells from a lethal

mutation in the RNase H gene The E coli [59] and

B subtilis [55] mutants that lack all RNase H genes

are not lethal, but show a temperature-sensitive growth

phenotype These results indicate that RNase H

activ-ity is dispensable for the growth of these

micro-organ-isms, but is involved in important cellular processes

We previously showed that two RNases H greatly

dif-fer in specific activities when they are simultaneously

produced in single cells and are therefore classified into

high- and low-activity type RNases H [37] These types

are not correlated with the RNase H families These

results suggest that bacteria evolve such that functional

redundancies of the RNase H genes are eliminated As

mentioned earlier, RNases H have been transferred

horizontally among different organisms An RNase H

transferred horizontally may provide a selective

advan-tage to recipients However, once a cell that already

has an RNase H receives a second RNase H by lateral

gene transfer, the responsibilities can be shared in ways

that would not necessarily be repeated following other

occurrences of transfer In some instances, the

incom-ing RNase H may retain the selective traits, whereas in

others, the resident and incoming RNase H may swap

some or all of their properties Because RNases HI

and HII represent high-activity type RNases H in

E coli and Shewanella sp SIB1, respectively, these

RNases H may retain the selective traits The

phylo-genetic and statistical analyses using 353 prokaryotic

genomes also suggest that functional redundancy

contributes to the exclusion or weakening of redundant

genes from the genome [60]

It is noted that not only the prokaryotic genomes,

but also the eukaryotic genomes usually contain

multiple RNase H genes This minireview is followed

others which focus on RNases H from eukaryotes

and retroviruses Table 1 summarizes large diversities

of RNases H in different organisms These organisms

are classified into several types based on differences

in the combination of the RNase H genes

Appar-ently, these types are not correlated with the species

of organisms

Perspectives

Prokaryotic RNases H vary greatly in domain struc-tures and substrate specificities For example, HBD-RNases HI and RNases HIII contain a substrate-binding domain at the N-termini Tko-RNase HII and Eco-RNase HI contain it at the C-terminus and middle

of the molecule, respectively Sto-RNase HI contains none of these domains Likewise, Sto-RNase HI cleaves the RNA strand of not only the RNA⁄ DNA hybrid, but also the RNA⁄ RNA duplex RNase HII can cleave a DNA⁄ DNA duplex containing a single ribonucleotide

at the DNA–RNA junction, whereas RNase HI and RNase HIII cannot Sto-RNase HI and Halo-RNase HI can cleave a RNA–DNA junction, although other RNases H cannot The RNase HII orthologue from T thermophilus can also cleave this junction, but cannot cleave a RNA⁄ DNA hybrid Understanding the substrate recognition mechanism of these RNases H with diverged structures and functions will allow us to answer the fundamental question how RNase H acquires its specificity for RNA⁄ DNA hybrids

Acknowledgements

We thank Drs H Chon, D J You, H Matsumura,

Y Koga and K Takano, for helpful discussions

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