Eukaryotic polymerases iota and zeta act sequentially to bypass DNA lesions

DNA lesions can often block DNA replication, so cells possess specialized low-fidelity, and often error-prone, DNA polymerases that can bypass such lesions and promote replication of damaged DNA. The Saccharomyces cerevisiae RAD30 and human hRAD30A encode Pol eta, which bypasses a cis-syn thymine-thymine dimer efficiently and accurately. Here we show that a related human gene, hRAD30B, encodes the DNA polymerase Pol iota, which misincorporates deoxynucleotides at a high rate. To bypass damage, Pol iota specifically incorporates deoxynucleotides opposite highly distorting or non-instructional DNA lesions. This action is combined with that of DNA polymerase Pol zeta, which is essential for damage-induced mutagenesis, to complete the lesion bypass. Pol zeta is very inefficient in inserting deoxynucleotides opposite DNA lesions, but readily extends from such deoxynucleotides once they have been inserted. Thus, in a new model for mutagenic bypass of DNA lesions in eukaryotes, the two DNA polymerases act sequentially: Pol iota incorporates deoxynucleotides opposite DNA lesions, and Pol zeta functions as a mispair extender.     

Single-molecule studies of the effect of template tension on T7 DNA polymerase activity

T7 DNA polymerase catalyses DNA replication in vitro at rates of more than 100 bases per second and has a 3'-->5' exonuclease (nucleotide removing) activity at a separate active site. This enzyme possesses a 'right hand' shape which is common to most polymerases with fingers, palm and thumb domains. The rate-limiting step for replication is thought to involve a conformational change between an 'open fingers' state in which the active site samples nucleotides, and a 'closed' state in which nucleotide incorporation occurs. DNA polymerase must function as a molecular motor converting chemical energy into mechanical force as it moves over the template. Here we show, using a single-molecule assay based on the differential elasticity of single-stranded and double-stranded DNA, that mechanical force is generated during the rate-limiting step and that the motor can work against a maximum template tension of approximately 34 pN. Estimates of the mechanical and entropic work done by the enzyme show that T7 DNA polymerase organizes two template bases in the polymerization site during each catalytic cycle. We also find a force-induced 100-fold increase in exonucleolysis above 40 pN.     

Error-prone replication of oxidatively damaged DNA by a high-fidelity DNA polymerase

Aerobic respiration generates reactive oxygen species that can damage guanine residues and lead to the production of 8-oxoguanine (8oxoG), the major mutagenic oxidative lesion in the genome. Oxidative damage is implicated in ageing and cancer, and its prevalence presents a constant challenge to DNA polymerases that ensure accurate transmission of genomic information. When these polymerases encounter 8oxoG, they frequently catalyse misincorporation of adenine in preference to accurate incorporation of cytosine. This results in the propagation of G to T transversions, which are commonly observed somatic mutations associated with human cancers. Here, we present sequential snapshots of a high-fidelity DNA polymerase during both accurate and mutagenic replication of 8oxoG. Comparison of these crystal structures reveals that 8oxoG induces an inversion of the mismatch recognition mechanisms that normally proofread DNA, such that the 8oxoG.adenine mismatch mimics a cognate base pair whereas the 8oxoG.cytosine base pair behaves as a mismatch. These studies reveal a fundamental mechanism of error-prone replication and show how 8oxoG, and DNA lesions in general, can form mismatches that evade polymerase error-detection mechanisms, potentially leading to the stable incorporation of lethal mutations.     

Structure of large fragment of Escherichia coli DNA polymerase I complexed with dTMP

The 3.3-A resolution crystal structure of the large proteolytic fragment of Escherichia coli DNA polymerase I complexed with deoxythymidine monophosphate consists of two domains, the smaller of which binds zinc-deoxythymidine monophosphate. The most striking feature of the larger domain is a deep crevice of the appropriate size and shape for binding double-stranded B-DNA. A flexible subdomain may allow the enzyme to surround completely the DNA substrate, thereby allowing processive nucleotide polymerization without enzyme dissociation.     

DNA primase acts as a molecular brake in DNA replication

A hallmark feature of DNA replication is the coordination between the continuous polymerization of nucleotides on the leading strand and the discontinuous synthesis of DNA on the lagging strand. This synchronization requires a precisely timed series of enzymatic steps that control the synthesis of an RNA primer, the recycling of the lagging-strand DNA polymerase, and the production of an Okazaki fragment. Primases synthesize RNA primers at a rate that is orders of magnitude lower than the rate of DNA synthesis by the DNA polymerases at the fork. Furthermore, the recycling of the lagging-strand DNA polymerase from a finished Okazaki fragment to a new primer is inherently slower than the rate of nucleotide polymerization. Different models have been put forward to explain how these slow enzymatic steps can take place at the lagging strand without losing coordination with the continuous and fast leading-strand synthesis. Nonetheless, a clear picture remains elusive. Here we use single-molecule techniques to study the kinetics of a multiprotein replication complex from bacteriophage T7 and to characterize the effect of primase activity on fork progression. We observe the synthesis of primers on the lagging strand to cause transient pausing of the highly processive leading-strand synthesis. In the presence of both leading- and lagging-strand synthesis, we observe the formation and release of a replication loop on the lagging strand. Before loop formation, the primase acts as a molecular brake and transiently halts progression of the replication fork. This observation suggests a mechanism that prevents leading-strand synthesis from outpacing lagging-strand synthesis during the slow enzymatic steps on the lagging strand.     

The XPV (xeroderma pigmentosum variant) gene encodes human DNA polymerase eta.

Xeroderma pigmentosum variant (XP-V) is an inherited disorder which is associated with increased incidence of sunlight-induced skin cancers. Unlike other xeroderma pigmentosum cells (belonging to groups XP-A to XP-G), XP-V cells carry out normal nucleotide-excision repair processes but are defective in their replication of ultraviolet-damaged DNA. It has been suspected for some time that the XPV gene encodes a protein that is involved in trans-lesion DNA synthesis, but the gene product has never been isolated. Using an improved cell-free assay for trans-lesion DNA synthesis, we have recently isolated a DNA polymerase from HeLa cells that continues replication on damaged DNA by bypassing ultraviolet-induced thymine dimers in XP-V cell extracts. Here we show that this polymerase is a human homologue of the yeast Rad30 protein, recently identified as DNA polymerase eta. This polymerase and yeast Rad30 are members of a family of damage-bypass replication proteins which comprises the Escherichia coli proteins UmuC and DinB and the yeast Rev1 protein. We found that all XP-V cells examined carry mutations in their DNA polymerase eta gene. Recombinant human DNA polymerase eta corrects the inability of XP-V cell extracts to carry out DNA replication by bypassing thymine dimers on damaged DNA. Together, these results indicate that DNA polymerase eta could be the XPV gene product.     

Requirement for the replication protein SSB in human DNA excision repair

Replication and repair are essential processes that maintain the continuity of the genetic material. Dissection of simian virus 40 (SV40) DNA replication has resulted in the identification of many eukaryotic replication proteins, but the biochemistry of the multienzyme process of DNA excision repair is less well defined. One protein that is absolutely required for semiconservative replication of SV40 DNA in vitro is human single-stranded DNA-binding protein (SSB, also called RF-A and RP-A). SSB consists of three polypeptides of relative molecular mass 70,000, 34,000 and 13,000, and acts with T antigen and topoisomerases to unwind DNA, allowing the access of other replication proteins. Human SSB can also stimulate the activity of polymerases alpha and delta, suggesting a further role in elongation during DNA replication. We have now found a role for human SSB in DNA excision repair using a cell-free system that can carry out nucleotide excision repair in vitro. Monoclonal antibodies against human SSB caused extensive inhibition of DNA repair in plasmid molecules damaged by ultraviolet light or acetylaminofluorene. Addition of purified SSB reversed this inhibition and further stimulated repair synthesis by increasing the number of repair events. These results show that a mammalian DNA replication protein is also essential for repair.     

Synthetic RNA-dependent DNA polymerase activity in normal rat liver and hepatomas

Normal adult rat liver contains a high level of a synthetic RNA-dependent DNA polymerase activity, which is distinct from cellular DNA-dependent polymerases. It uses poly(dT).poly(rA) and poly(rA).poly(rU) as templates but has little or no response to DNA or several single-stranded RNAs.     

耐热性重组Taq DNA酶的制备及鉴定

利用含Taq DNA聚合酶基因的pTaq表达质粒转化大肠杆菌E.coli菌株,用异丙基硫代-β-D-乳糖苷(IPTG)诱导10～12h表达耐热Taq DNA聚合酶;然后溶菌酶、NP40裂解细菌;硫酸铵沉淀、4℃下透析;SDS-聚丙烯酰胺凝胶电泳(SDS-PAGE)、考马斯亮蓝法和PCR分析其纯度、浓度和活性。结果表明分离纯化制备的耐热性Taq DNA聚合酶,其纯度、浓度和活性均可与同类产品相比,比活性达20000U/mg,能有效扩增出DNA片段。     

RecA acts in trans to allow replication of damaged DNA by DNA polymerase V

The DNA polymerase V (pol V) and RecA proteins are essential components of a mutagenic translesion synthesis pathway in Escherichia coli designed to cope with DNA damage. Previously, it has been assumed that RecA binds to the DNA template strand being copied. Here we show, however, that pol-V-catalysed translesion synthesis, in the presence or absence of the beta-processivity-clamp, occurs only when RecA nucleoprotein filaments assemble or RecA protomers bind on separate single-stranded (ss)DNA molecules in trans. A 3'-proximal RecA filament end on trans DNA is essential for stimulation; however, synthesis is strengthened by further pol V-RecA interactions occurring elsewhere along a trans nucleoprotein filament. We suggest that trans-stimulation of pol V by RecA bound to ssDNA reflects a distinctive regulatory mechanism of mutation that resolves the paradox of RecA filaments assembled in cis on a damaged template strand obstructing translesion DNA synthesis despite the absolute requirement of RecA for SOS mutagenesis.     

Preferential cis-syn thymine dimer bypass by DNA polymerase eta occurs with biased fidelity

Human DNA polymerase eta (Pol eta) modulates susceptibility to skin cancer by promoting DNA synthesis past sunlight-induced cyclobutane pyrimidine dimers that escape nucleotide excision repair (NER). Here we have determined the efficiency and fidelity of dimer bypass. We show that Pol eta copies thymine dimers and the flanking bases with higher processivity than it copies undamaged DNA, and then switches to less processive synthesis. This ability of Pol eta to sense the dimer location as synthesis proceeds may facilitate polymerase switching before and after lesion bypass. Pol eta bypasses a dimer with low fidelity and with higher error rates at the 3' thymine than at the 5' thymine. A similar bias is seen with Sulfolobus solfataricus DNA polymerase 4, which forms a Watson-Crick base pair at the 3' thymine of a dimer but a Hoogsteen base pair at the 5' thymine (ref. 3). Ultraviolet-induced mutagenesis is also higher at the 3' base of dipyrimidine sequences. Thus, in normal people and particularly in individuals with NER-defective xeroderma pigmentosum who accumulate dimers, errors made by Pol eta during dimer bypass could contribute to mutagenesis and skin cancer.     

Stimulation of protein-directed strand exchange by a DNA helicase

The protein-mediated exchange of strands between a DNA double helix and a homologous DNA single strand involves both synapsis and branch migration, which are two important aspects of any general recombination reaction. Purified DNA-dependent ATPases from Escherichia coli (recA protein), Ustilago (rec 1 protein) and phage T4 (uvsX protein) have been shown to drive both synapsis and branch migration in vitro. The T4 gene 32 protein is a helix-destabilizing protein that greatly stimulates uvsX-protein-catalysed synapsis, and the E. coli SSB (single-strand binding) protein stimulates the analogous recA-protein-mediated reaction to a lesser degree. One suspects that several other proteins also play a role in the strand exchange process. For example, a DNA helicase could in principle accelerate branch migration rates by helping to melt the helix at the branch point. The T4 dda protein is a DNA helicase that is required to move the T4 replication fork past DNA template-bound proteins in vitro. Previously, we have shown that the dda protein binds to a column that contains immobilized T4 uvsX protein. We show here that this helicase specifically stimulates the branch migration reaction that the uvsX protein catalyses as a central part of the genetic recombination process in a T4 bacteriophage-infected cell.