Exonucleolytic Activity Research Articles

Exonucleolysis is required for nuclear mRNA quality control in yeast THO mutants

Production of aberrant messenger ribonucleoprotein particles (mRNPs) is subject to quality control (QC). In yeast strains carrying mutations of the THO complex, transcription induction triggers a number of interconnected QC phenotypes: (1) rapid degradation of several mRNAs; (2) retention of a fraction of THO-dependent mRNAs in transcription site-associated foci; and (3) formation of a high molecular weight DNA/protein complex in the 3'-ends of THO target genes. Here, we demonstrate that the 3'-5' exonucleolytic domain of the nuclear exosome factor Rrp6p is necessary for establishing all QC phenotypes associated with THO mutations. The N terminus of Rrp6p is also important presumably through its binding to the Rrp6p co-factor Rrp47p. Interestingly, the 3'-5' exonucleolytic activity of Dis3p, the only other active exonuclease of the nuclear exosome, can also contribute to RNA QC in THO mutants, while other nuclear 3'-5' exonucleases cannot. Our data show that exonucleolytic attack by the nuclear exosome is needed both for provoking mRNP QC and for its ensuing elimination of faulty RNA.

Significance of Stereochemistry of 3′-Terminal Phosphorothioate-modified Primer in DNA Polymerase-mediated Chain Extension

Influence of stereochemistry of the 3'-terminal phosphorothioate (PS)-modified primers was studied in a single base extension (SBE) assay to evaluate any improvements in specificity. SBE reactions were catalyzed by members of the high fidelity Pfu family of DNA polymerases with (exo+) or without (exo-) 3' --> 5' exonucleolytic activity. The diastereomerically pure PS-labeled primers used in these studies were obtained either by the stereospecific chemical synthesis invented in our laboratory or by the more conventional ion-exchange chromatographic method for separation of a mixture of diastereomers (R(P) and S(P)). When the SBE reaction was performed in the presence of mispaired 2'-deoxyribonucleoside triphosphates (dNTPs), the "racemic" 3'-phosphorothioate primer mixture resulted in a lower level of 3' --> 5' exonuclease-mediated cleavage products in comparison to the SBE reactions carried out with the corresponding unmodified primer. When the diastereomerically pure RP 3'-phosphorothioate primer was examined, the results were largely the same as for the racemic 3'-phosphorothioate primer mixture. In contrast, a 3'-PS primer of S(P) configuration displayed significantly improved performance in the SBE reaction. This included the lack of 3' --> 5' proofreading products, less mispriming, and improved yield of incorporation of the correct nucleotide.

Endonucleolytic mutation analysis by internal labeling (EMAIL)

Mismatch-specific endonucleases are efficient tools for the targeted scanning of populations for subtle DNA variations. Conventional protocols involve 5'-labeled amplicon substrates and the detection of digestion products by LIF electrophoresis. A shortcoming of such protocols, however, is the limited 5'-signal strength. Normally the sensitivity of fluorescent DNA analyzers is superior to that of intercalating dye/agarose systems, however, pooling capacities of the former and latter approaches to mismatch scanning are somewhat similar. Detection is further limited by significant background. We investigated the activity of CEL nucleases using amplicon substrates labeled both internally and at each 5'-terminus. The amplicons were generated from exon 8 of the rice starch synthase IIa encoding gene. Signal of both 5'-labels was significantly reduced by enzyme activity, while that of the internal label was largely unaffected. In addition, background resulting from internal labeling was a significant improvement on that associated with 5'-labeling. Sizing of the multilabeled substrates suggests that 5'-modification enhances exonucleolytic activity, resulting in the removal of the dye-labeled terminal nucleotides. We have developed an alternative approach to mismatch detection, in which amplicon labeling is achieved via the incorporation of fluorescently labeled deoxynucleotides, which we have named Endonucleolytic Mutation Analysis by Internal Labeling (EMAIL). The strength of the EMAIL assay was demonstrated by the reclassification of a rice line as being heterozygous for the starch gene. This cultivar was assigned as being homozygous by a previous resequencing study. EMAIL shows potential for the clear identification of multiple mutations amongst allelic pools.

Exosome-mediated quality control: Substrate recruitment and molecular activity

The eukaryotic exosome is a multisubunit complex that is mainly responsible for 3'-5' exonucleolytic degradation of RNAs, both in the nucleus and the cytoplasm. In this review we summarize the recent experiments that have provided information on the organisation, structure and activity of this large assembly. Interestingly, eukaryotic exosomes have been implicated in a large number of RNA degradation pathways including recently discovered RNA quality control mechanisms. A variety of cofactors have been shown to participate in substrate recruitment and/or assist exonucleolytic activities. Despite this avalanche of new results, further analyses will be required to improve our understanding of exosome regulation.

Duality of polynucleotide substrates for Phi29 DNA polymerase: 3′→5′ RNase activity of the enzyme

Phi29 DNA polymerase is a small DNA-dependent DNA polymerase that belongs to eukaryotic B-type DNA polymerases. Despite the small size, the polymerase is a multifunctional proofreading-proficient enzyme. It catalyzes two synthetic reactions (polymerization and deoxynucleotidylation of Phi29 terminal protein) and possesses two degradative activities (pyrophosphorolytic and 3'-->5' DNA exonucleolytic activities). Here we report that Phi29 DNA polymerase exonucleolyticaly degrades ssRNA. The RNase activity acts in a 3' to 5' polarity. Alanine replacements in conserved exonucleolytic site (D12A/D66A) inactivated RNase activity of the enzyme, suggesting that a single active site is responsible for cleavage of both substrates: DNA and RNA. However, the efficiency of RNA hydrolysis is approximately 10-fold lower than for DNA. Phi29 DNA polymerase is widely used in rolling circle amplification (RCA) experiments. We demonstrate that exoribonuclease activity of the enzyme can be used for the target RNA conversion into a primer for RCA, thus expanding application potential of this multifunctional enzyme and opening new opportunities for RNA detection.

The NF-κB Canonical Pathway Is Involved in the Control of the Exonucleolytic Processing of Coding Ends during V(D)J Recombination

V(D)J recombination is essential to produce an Ig repertoire with a large range of Ag specificities. Although NF-kappaB-binding sites are present in the human and mouse IgH, Igkappa, and Iglambda enhancer modules and RAG expression is controlled by NF-kappaB, it is not known whether NF-kappaB regulates V(D)J recombination mechanisms after RAG-mediated dsDNA breaks. To clarify the involvement of NF-kappaB in human V(D)J recombination, we amplified Ig gene rearrangements from individual peripheral B cells of patients with X-linked anhidrotic ectodermal dysplasia with hyper-IgM syndrome (HED-ID) who have deficient expression of the NF-kappaB essential modulator (NEMO/Ikkgamma). The amplification of nonproductive Ig gene rearrangements from HED-ID B cells reflects the influence of the Ikkgamma-mediated canonical NF-kappaB pathway on specific molecular mechanisms involved in V(D)J recombination. We found that the CDR3(H) from HED-ID B cells were abnormally long, as a result of a marked reduction in the exonuclease activity on the V, D, and J germline coding ends, whereas random N-nucleotide addition and palindromic overhangs (P nucleotides) were comparable to controls. This suggests that an intact canonical NF-kappaB pathway is essential for normal exonucleolytic activity during human V(D)J recombination, whereas terminal deoxynucleotide transferase, Artemis, and DNA-dependent protein kinase catalytic subunit activity are not affected. The generation of memory B cells and somatic hypermutation were markedly deficient confirming a role for NF-kappaB in these events of B cell maturation. However, selection of the primary B cell repertoire appeared to be intact and was partially able to correct the defects generated by abnormal V(D)J recombination.

The Characterization of Saccharomyces cerevisiae Mre11/Rad50/Xrs2 Complex Reveals that Rad50 Negatively Regulates Mre11 Endonucleolytic but not the Exonucleolytic Activity

The evolutionarily conserved heterotrimeric Mre11/Rad50/Xrs2 (Nbs1) (MRX/N) complex plays a central role in an array of cellular responses involving DNA damage, telomere length homeostasis, cell-cycle checkpoint control and meiotic recombination. The underlying biochemical functions of MRX/N complex, or each of its individual subunits, at telomeres and the importance of complex formation are poorly understood. Here, we show that the Saccharomyces cerevisiae MRX complex, or its subunits, display an overwhelming preference for G-quadruplex DNA than for telomeric single-stranded or double-stranded DNA implicating the possible existence of this DNA structure in vivo. Although these alternative DNA substrates failed to affect Rad50 ATPase activity, kinetic analyses revealed that interaction of Rad50 with Xrs2 and/or Mre11 led to a twofold increase in the rates of ATP hydrolysis. Significantly, we show that Mre11 displays sequence-specific double-stranded DNA endonuclease activity, and Rad50, but not Xrs2, abrogated endonucleolytic but not the exonucleolytic activity. This repression was alleviated upon ATP hydrolysis by Rad50, suggesting that complex formation between Rad50 and Mre11 might be important for blocking the inappropriate cleavage of genomic DNA. Mre11 alone, or in the presence of ATP, MRX, MR or MX sub-complexes cleaved at the 5′ end of an array of G residues in single-stranded DNA, at G quartets in G4 DNA, and at the center of TGTG repeats in duplex DNA. We propose that negative regulation of Mre11 endonuclease activity by Rad50 might be important for native as well as de novo telomere length homeostasis.

A mutation in TREX1 that impairs susceptibility to granzyme A-mediated cell death underlies familial chilblain lupus

We recently described a novel autosomal-dominant genodermatosis, termed familial chilblain lupus, and mapped its genetic locus to chromosome 3p21. Familial chilblain lupus manifests in early childhood with ulcerating acral skin lesions and is associated with arthralgias and circulating antinuclear antibodies. In this study, we report the identification of a heterozygous missense mutation (D18N) in TREX1 encoding the 3'-5'repair exonuclease 1 in affected individuals of the family with chilblain lupus. The homodimeric TREX1 is the most abundant intracellular DNase in mammalian cells. We have recently shown that TREX1 plays a role in apoptotic single-stranded DNA damage induced by the killer lymphocyte protease granzyme A. D18N affects a highly conserved amino acid residue critical for catalytic activity. Recombinant mutant TREX1 homodimers are enzymatically inactive, while wild type/mutant heterodimers show residual exonucleolytic activity, suggesting a heterozygous loss of function. Lymphoblastoid cells carrying the D18N mutation are significantly less sensitive to granzyme A-mediated cell death, suggesting a novel role for this caspase-independent form of apoptosis in the pathogenesis of familial chilblain lupus. Our findings also warrant further investigation of TREX1 in common forms of lupus erythematosus.

Dissecting mechanisms of nuclear mRNA surveillance in THO/sub2 complex mutants

The nuclear exosome is involved in numerous RNA metabolic processes. Exosome degradation of rRNA, snoRNA, snRNA and tRNA in Saccharomyces cerevisiae is activated by TRAMP complexes, containing either the Trf4p or Trf5p poly(A) polymerase. These enzymes are presumed to facilitate exosome access by appending oligo(A)-tails onto structured substrates. Another role of the nuclear exosome is that of mRNA surveillance. In strains harboring a mutated THO/Sub2p system, involved in messenger ribonucleoprotein particle biogenesis and nuclear export, the exosome-associated 3' --> 5' exonuclease Rrp6p is required for both retention and degradation of nuclear restricted mRNAs. We show here that Trf4p, in the context of TRAMP, is an mRNA surveillance factor. However, unlike Rrp6p, Trf4p only partakes in RNA degradation and not in transcript retention. Surprisingly, a polyadenylation-defective Trf4p protein is fully active, suggesting polyadenylation-independent mRNA degradation. Transcription pulse-chase experiments show that HSP104 molecules undergoing quality control in THO/sub2 mutant strains fall into two distinct populations: One that is quickly degraded after transcription induction and another that escapes rapid decay and accumulates in foci associated with the HSP104 transcription site.

The role of the S1 domain in exoribonucleolytic activity: Substrate specificity and multimerization

RNase II is a 3'-5' exoribonuclease that processively hydrolyzes single-stranded RNA generating 5' mononucleotides. This enzyme contains a catalytic core that is surrounded by three RNA-binding domains. At its C terminus, there is a typical S1 domain that has been shown to be critical for RNA binding. The S1 domain is also present in the other major 3'-5' exoribonucleases from Escherichia coli: RNase R and polynucleotide phosphorylase (PNPase). In this report, we examined the involvement of the S1 domain in the different abilities of these three enzymes to overcome RNA secondary structures during degradation. Hybrid proteins were constructed by replacing the S1 domain of RNase II for the S1 from RNase R and PNPase, and their exonucleolytic activity and RNA-binding ability were examined. The results revealed that both the S1 domains of RNase R and PNPase are able to partially reverse the drop of RNA-binding ability and exonucleolytic activity resulting from removal of the S1 domain of RNase II. Moreover, the S1 domains investigated are not equivalent. Furthermore, we demonstrate that S1 is neither responsible for the ability to overcome secondary structures during RNA degradation, nor is it related to the size of the final product generated by each enzyme. In addition, we show that the S1 domain from PNPase is able to induce the trimerization of the RNaseII-PNP hybrid protein, indicating that this domain can have a role in the biogenesis of multimers.

A phosphate‐binding subsite in bovine pancreatic ribonuclease A can be converted into a very efficient catalytic site

A general acid-base catalytic mechanism is responsible for the cleavage of the phosphodiester bonds of the RNA by ribonuclease A (RNase A). The main active site is formed by the amino acid residues His12, His119, and Lys41, and the process follows an endonucleolytic pattern that depends on the existence of a noncatalytic phosphate-binding subsite adjacent, on the 3'-side, to the active site; in this region the phosphate group of the substrate establishes electrostatic interactions through the side chains of Lys7 and Arg10. We have obtained, by means of site-directed mutagenesis, RNase A variants with His residues both at positions 7 and 10. These mutations have been introduced with the aim of transforming a noncatalytic binding subsite into a putative new catalytic active site. The RNase activity of these variants was determined by the zymogram technique and steady-state kinetic parameters were obtained by spectrophotometric methods. The variants showed a catalytic efficiency in the same order of magnitude as the wild-type enzyme. However, we have demonstrated in these variants important effects on the substrate's cleavage pattern. The quadruple mutant K7H/R10H/H12K/H119Q shows a clear increase of the exonucleolytic activity; in this case the original native active site has been suppressed, and, as consequence, its activity can only be associated to the new active site. In addition, the mutant K7H/R10H, with two putative active sites, also shows an increase in the exonucleolytic preference with respect to the wild type, a fact that may be correlated with the contribution of the new active site.

Identification of an mRNA-Decapping Regulator Implicated in X-Linked Mental Retardation

Two major decapping enzymes are involved in the decay of eukaryotic mRNA, Dcp2 and DcpS. Despite the detection of robust DcpS decapping activity in cell extract, minimal to no decapping is detected from human Dcp2 (hDcp2) in extract. We now demonstrate that one reason for the lack of detectable hDcp2 activity in extract is due to the presence of inhibitory trans factor(s). Furthermore, we demonstrate that a previously identified testis-specific protein of unknown function implicated in nonspecific X-linked mental retardation, VCX-A, can function as an inhibitor of hDcp2 decapping in vitro and in cells. VCX-A is a noncanonical cap-binding protein that binds to capped RNA but not cap structure lacking an RNA. Its cap association is enhanced by hDcp2 to further augment the ability of VCX-A to inhibit decapping. Our data demonstrate that VCX-A can regulate mRNA stability and that it is an example of a tissue-specific decapping regulator.

A unique error signature for human DNA polymerase ν

Human DNA polymerase ν (pol ν) is one of three A family polymerases conserved in vertebrates. Although its biological functions are unknown, pol ν has been implicated in DNA repair and in translesion DNA synthesis (TLS). Pol ν lacks intrinsic exonucleolytic proofreading activity and discriminates poorly against misinsertion of dNTP opposite template thymine or guanine, implying that it should copy DNA with low base substitution fidelity. To test this prediction and to comprehensively examine pol ν DNA synthesis fidelity as a clue to its function, here we describe human pol ν error rates for all 12 single base–base mismatches and for insertion and deletion errors during synthesis to copy the lacZ α-complementation sequence in M13mp2 DNA. Pol ν copies this DNA with average single-base insertion and deletion error rates of 7 × 10 −5 and 17 × 10 −5, respectively. This accuracy is comparable to that of replicative polymerases in the B family, lower than that of its A family homolog, human pol γ, and much higher than that of Y family TLS polymerases. In contrast, the average single-base substitution error rate of human pol ν is 3.5 × 10 −3, which is inaccurate compared to the replicative polymerases and comparable to Y family polymerases. Interestingly, the vast majority of errors made by pol ν reflect stable misincorporation of dTMP opposite template G, at average rates that are much higher than for homologous A family members. This pol ν error is especially prevalent in sequence contexts wherein the template G is preceded by a C–G or G–C base pair, where error rates can exceed 10%. Amino acid sequence alignments based on the structures of more accurate A family polymerases suggest substantial differences in the O-helix of pol ν that could contribute to this unique error signature.

DNA Degradation at Unprotected Telomeres in Yeast Is Regulated by the CDK1 (Cdc28/Clb) Cell-Cycle Kinase

In the absence of functional telomeric cap protection, the ends of eukaryotic chromosomes are subject to DNA damage responses that lead to cell-cycle arrest and, eventually, genomic instability. However, the controlling activities responsible for the initiation of genome instability on unprotected telomeres remained unclear. Here we show that in budding yeast, unprotected telomeres undergo a tightly cell-cycle-regulated DNA degradation. Ablation of the function of essential capping proteins Cdc13p or Stn1p only caused telomere degradation in G2/M, but not in G1 of the cell cycle. Accordingly, G1-arrested cells with unprotected telomeres remained viable, while G2/M-arrested cells failed to recover. The data also show that completion of S phase and the activity of the S-Cdk1 kinase were required for telomere degradation. These results strongly suggest that after a loss of the telomere capping function, telomere-led genome instability is caused by tightly regulated cellular DNA repair attempts.

Structure of the Escherichia coli DNA polymerase III epsilon-HOT proofreading complex.

The epsilon subunit of Escherichia coli DNA polymerase III possesses 3'-exonucleolytic proofreading activity. Within the Pol III core, epsilon is tightly bound between the alpha subunit (DNA polymerase) and subunit. Here, we present the crystal structure of epsilon in complex with HOT, the bacteriophage P1-encoded homolog of , at 2.1 A resolution. The epsilon-HOT interface is defined by two areas of contact: an interaction of the previously unstructured N terminus of HOT with an edge of the epsilon central beta-sheet as well as interactions between HOT and the catalytically important helix alpha1-loop-helix alpha2 motif of epsilon. This structure provides insight into how HOT and, by implication, may stabilize the epsilon subunit, thus promoting efficient proofreading during chromosomal replication.

Integrating snoRNP assembly with mRNA biogenesis

The biogenesis of translatable eukaryotic messenger RNA (mRNA) requires a series of post‐transcriptional processing steps. Initially, the 5′‐end of the nascent pre‐mRNA acquires a cap structure. Then, the intron regions are successively spliced out from the growing RNA chain. Finally, the mRNA 3′‐end is cleaved and a poly(A) tail is added. To coordinate efficient and faithful mRNA production, a complex network of dynamic physical and functional interactions are formed between the elongating RNA polymerase (pol) II and the different mRNA‐processing machines that carry out each of these steps (Ares & Proudfoot, 2005). In vertebrates, the intron regions of protein‐coding genes frequently encode small‐nucleolar RNAs (snoRNAs). These snoRNAs are post‐transcriptionally processed from the spliced out and debranched host intron by exonucleolytic activities. It has long been assumed that the only role of the RNA pol II transcription/splicing machine in intronic snoRNA expression is to provide the linear substrate RNA for the processing exonucleases. However, recent studies from several laboratories have revealed more intimate functional interplays between the machines that are devoted to snoRNA processing and mRNA biogenesis. The intronic snoRNAs fall into two structurally and functionally well‐defined classes. SnoRNAs carrying the conserved box C and D elements function mainly as guides in the site‐specific 2′‐ O ‐methylation of ribosomal RNAs (rRNAs), whereas box H/ACA snoRNAs direct the pseudouridylation of rRNAs (Terns & Terns, 2002). To form …

Discovery of an RNA virus 3′→5′ exoribonuclease that is critically involved in coronavirus RNA synthesis

Replication of the giant RNA genome of severe acute respiratory syndrome (SARS) coronavirus (CoV) and synthesis of as many as eight subgenomic (sg) mRNAs are mediated by a viral replicase-transcriptase of outstanding complexity that includes an essential endoribonuclease activity. Here, we show that the CoV replicative machinery, unlike that of other RNA viruses, also uses an exoribonuclease (ExoN) activity, which is associated with nonstructural protein (nsp) 14. Bacterially expressed forms of SARS-CoV nsp14 were shown to act on both ssRNAs and dsRNAs in a 3'-->5' direction. The activity depended on residues that are conserved in the DEDD exonuclease superfamily. The protein did not hydrolyze DNA or ribose-2'-O-methylated RNA substrates and required divalent metal ions for activity. A range of 5'-labeled ssRNA substrates were processed to final products of approximately 8-12 nucleotides. When part of dsRNA or in the presence of nonlabeled dsRNA, the 5'-labeled RNA substrates were processed to significantly smaller products, indicating that binding to dsRNA in cis or trans modulates the exonucleolytic activity of nsp14. Characterization of human CoV 229E ExoN active-site mutants revealed severe defects in viral RNA synthesis, and no viable virus could be recovered. Besides strongly reduced genome replication, specific defects in sg RNA synthesis, such as aberrant sizes of specific sg RNAs and changes in the molar ratios between individual sg RNA species, were observed. Taken together, the study identifies an RNA virus ExoN activity that is involved in the synthesis of multiple RNAs from the exceptionally large genomic RNA templates of CoVs.

Eukaryotic error-prone DNA polymerases: The presumed roles in replication, repair, and mutagenesis

A number of error-prone DNA polymerases is found among eukaryotes from yeasts up to mammalia including humans. According to the partial homology of a primary structure, they are united in families B, X, Y and display high infidelity on uninjured DNA-template, whereas they are rather accurate on DNA injuries. These DNA polymerases are characterized by the probability of base substitutions or frame shifts of 10(-3) to 7.5 x 10(-1) on DNA injuries, whereas the probability of spontaneous mutagenesis per replicated nucleotide accounts 10(-10) - 10(-12). Inaccurate DNA polymerases are terminal deoxynucleotidyl transferase (TdT), DNA polymerases beta, zeta, kappa, eta, iota, lamda, mu, and Rev1. Their principal properties are described in this review. All of the polymerases under study are deprived of the corrective 3'-->5' exonucleolytic activity. The specialization of these polymerases is contained in the capability to synthesize opposite DNA lesions (not eliminated by multiple repair systems) that is explained by the flexibility of their active sites or by the limited capability to exhibit the TdT activity. Classic DNA polymerases alpha, delta, epsilon, and gamma cannot elongate the primers with mismatched nucleotides on their 3'-ends (that leads to the replication block), whereas some of the specialized polymerases can do it. It is accompanied by the overcoming of a replication block, often with the expense of an elevated mutagenesis. How can a cell live under the conditions of such a huge infidelity of many DNA polymerases? Error-prone DNA polymerases are not found in all tissues though some of them are essential for an organism survival. Furthermore, cells must not allow for these polymerases to work effectively on uninjured DNA. After bypass of a lesion on DNA-template, it is necessary, as soon as possible, to switch catalysis of the DNA synthesis from the specialized polymerases on the relatively accurate DNA polymerases delta and epsilon (fidelity of 10(-5) - 10(-6)). It is made by the formation of the complexes of polymerases delta or epsilon with PCNA and replicative factors RP-A and RF-C. Such highly processive complexes manifest the bigger affinity to the correct primers than the specialized DNA polymerases do. The switching is stimulated by distributivity or weak processivity of the specialized DNA polymerases. The accuracy of these polymerases are augmented by the action of the corrective 3'-exonucleolytic function of DNA polymerases delta and epsilon as well as by the autonomous 3'-->5' exonucleases which are widespread among the representatives of the whole phylogenetic tree. Exonucleolytic correction slows down the replication in the presence of lesions in DNA-template but makes the replication more accurate that decreases the probability of mutagenesis and carcinogenesis.

The mitochondrial theory of aging and its relationship to reactive oxygen species damage and somatic mtDNA mutations.

Mitochondria are cellular energy factories that generate ATP via the reaction of hydrocarbons with oxygen. Every human cell contains hundreds of mitochondria, and each mitochondrion contains multiple copies of mitochondrial DNA (mtDNA). The ancestry of the mitochondrial genome can be traced to early eubacteria, and it is therefore unexpected that this organelle may have a major role in governing the pace of human aging. Three recent papers (1–3) plus a work published in a recent issue of PNAS (4) have demonstrated that accelerating the mtDNA mutation rate can result in some features suggestive of premature aging, consistent with the view that loss of mitochondrial function is a major causal factor in aging. mtDNA polymerase (Pol-γ) is the only DNA polymerase that is known to be targeted to and that resides in mitochondria (5). In the absence of other known mtDNA polymerases, it is assumed that Pol-γ is responsible for all aspects of mtDNA synthesis, including replication of the mitochondrial genome and repair of DNA damage. As is the case for many other DNA polymerases, the high fidelity of Pol-γ derives from both selection for the correct incoming nucleotide and excision of misincorporated nucleotides by a 3′ → 5′ exonucleolytic proofreading activity (6). Elimination of proofreading by replacement of critical aspartic acid residues in the exonuclease domain with alanine increases misincorporation; in vitro , this misincorporation is manifested predominantly as single base substitutions (7). Both the Larsson (1) and Prolla (2) groups replaced in embryonic stem cells POLG, the …

Bacillus subtilis Bacteriophage SPP1-encoded Gene 34.1 Product is a Recombination-dependent DNA Replication Protein

SPP1-encoded replication and recombination proteins, involved in the early steps of the initiation of concatemeric DNA synthesis, have been analyzed. Dimeric G34.1P exonuclease degrades, with a 5' to 3' polarity and in a Mg2+-dependent reaction, preferentially linear double-stranded (ds) DNA rather than single-stranded (ss) DNA. Binding of the replisome organizer, G38P, to its cognate sites (oriDNA) halts the 5' to 3' exonucleolytic activity of G34.1P on dsDNA. The G35P recombinase increases the affinity of G34.1P for dsDNA, and stimulates G34.1P activity on dsDNA, but not on ssDNA. Then, filamented G35P promotes limited strand exchange with a homologous sequence. The ssDNA binding protein, G36P, protects ssDNA from the G34.1P exonuclease activity and stimulates G35P-catalyzed strand exchange. The data presented suggest a model for the role of G34.1P during initiation of sigma replication: G38P bound to oriDNA might halt replication fork progression, and G35P, G34.1P and G36P in concert might lead to the re-establishment of a unidirectional recombination-dependent replication that accounts for the direction of DNA packaging.