DNA polymerases: Hoogsteen base-pairing in DNA replication?

Human polymerase-iota belongs to the error-prone Y family of polymerases, which frequently incorporate incorrect nucleotides during DNA replication but can efficiently bypass DNA lesions. On the basis of X-ray diffraction data, Nair et al. propose that Hoogsteen base-pairing is adopted by DNA during its replication by this enzyme. Here I re-examine their X-ray data and find that the electron density is very weak for a Hoogsteen base pair formed between a template adenine deoxyribonucleotide in the syn conformation and a deoxythymidine 5'-triphosphate (dTTP), and that the fit is better for a normal Watson-Crick base pair. As a guanine-cytosine (G-C) base pair has no potential to form a Hoogsteen base pair at physiological pH, Hoogsteen base-pairing is unlikely to be used in replication by this polymerase.     

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.     

A specific partner for abasic damage in DNA.

In most models of DNA replication, Watson-Crick hydrogen bonding drives the incorporation of nucleotides into the new strand of DNA and maintains the complementarity of bases with the template strand. Studies with nonpolar analogues of thymine and adenine, however, have shown that replication is still efficient in the absence of hydrogen bonds. The replication of base pairs might also be influenced by steric exclusion, whereby inserted nucleotides need to be the correct size and shape to fit the active site against a template base. A simple steric-exclusion model may not require Watson-Crick hydrogen bonding to explain the fidelity of replication, nor should canonical purine and pyrimidine shapes be necessary for enzymatic synthesis of a base pair if each can fit into the DNA double helix without steric strain. Here we test this idea by using a pyrene nucleoside triphosphate (dPTP) in which the fluorescent 'base' is nearly as large as an entire Watson-Crick base pair. We show that the non-hydrogen-bonding dPTP is efficiently and specifically inserted by DNA polymerases opposite sites that lack DNA bases. The efficiency of this process approaches that of a natural base pair and the specificity is 10(2)-10(4)-fold. We use these properties to sequence abasic lesions in DNA, which are a common form of DNA damage in vivo. In addition to their application in identifying such genetic lesions, our results show that neither hydrogen bonds nor purine and pyrimidine structures are required to form a base pair with high efficiency and selectivity. These findings confirm that steric complementarity is an important factor in the fidelity of DNA synthesis.     

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.     

8-oxo-guanine bypass by human DNA polymerases in the presence of auxiliary proteins

Specialized DNA polymerases (DNA pols) are required for lesion bypass in human cells. Auxiliary factors have an important, but so far poorly understood, role. Here we analyse the effects of human proliferating cell nuclear antigen (PCNA) and replication protein A (RP-A) on six different human DNA pols--belonging to the B, Y and X classes--during in vitro bypass of different lesions. The mutagenic lesion 8-oxo-guanine (8-oxo-G) has high miscoding potential. A major and specific effect was found for 8-oxo-G bypass with DNA pols lambda and eta. PCNA and RP-A allowed correct incorporation of dCTP opposite a 8-oxo-G template 1,200-fold more efficiently than the incorrect dATP by DNA pol lambda, and 68-fold by DNA pol eta, respectively. Experiments with DNA-pol-lambda-null cell extracts suggested an important role for DNA pol lambda. On the other hand, DNA pol iota, together with DNA pols alpha, delta and beta, showed a much lower correct bypass efficiency. Our findings show the existence of an accurate mechanism to reduce the deleterious consequences of oxidative damage and, in addition, point to an important role for PCNA and RP-A in determining a functional hierarchy among different DNA pols in lesion bypass.     

Structural basis for template-independent RNA polymerization

The 3'-terminal CCA nucleotide sequence (positions 74-76) of transfer RNA is essential for amino acid attachment and interaction with the ribosome during protein synthesis. The CCA sequence is synthesized de novo and/or repaired by a template-independent RNA polymerase, 'CCA-adding enzyme', using CTP and ATP as substrates. Despite structural and biochemical studies, the mechanism by which the CCA-adding enzyme synthesizes the defined sequence without a nucleic acid template remains elusive. Here we present the crystal structure of Aquifex aeolicus CCA-adding enzyme, bound to a primer tRNA lacking the terminal adenosine and an incoming ATP analogue, at 2.8 A resolution. The enzyme enfolds the acceptor T helix of the tRNA molecule. In the catalytic pocket, C75 is adjacent to ATP, and their base moieties are stacked. The complementary pocket for recognizing C74-C75 of tRNA forms a 'protein template' for the penultimate two nucleotides, mimicking the nucleotide template used by template-dependent polymerases. These results are supported by systematic analyses of mutants. Our structure represents the 'pre-insertion' stage of selecting the incoming nucleotide and provides the structural basis for the mechanism underlying template-independent RNA polymerization.     

Replication of a cis-syn thymine dimer at atomic resolution

Ultraviolet light damages DNA by catalysing covalent bond formation between adjacent pyrimidines, generating cis-syn cyclobutane pyrimidine dimers (CPDs) as the most common lesion. CPDs block DNA replication by high-fidelity DNA polymerases, but they can be efficiently bypassed by the Y-family DNA polymerase pol eta. Mutations in POLH encoding pol eta are implicated in nearly 20% of xeroderma pigmentosum, a human disease characterized by extreme sensitivity to sunlight and predisposition to skin cancer. Here we have determined two crystal structures of Dpo4, an archaeal pol eta homologue, complexed with CPD-containing DNA, where the 3' and 5' thymine of the CPD separately serves as a templating base. The 3' thymine of the CPD forms a Watson-Crick base pair with the incoming dideoxyATP, but the 5' thymine forms a Hoogsteen base pair with the dideoxyATP in syn conformation. Dpo4 retains a similar tertiary structure, but each unusual DNA structure is individually fitted into the active site for catalysis. A model of the pol eta-CPD complex built from the crystal structures of Saccharomyces cerevisiae apo-pol eta and the Dpo4-CPD complex suggests unique features that allow pol eta to efficiently bypass CPDs.     

MutT protein specifically hydrolyses a potent mutagenic substrate for DNA synthesis.

Errors in the replication of DNA are a major source of spontaneous mutations, and a number of cellular functions are involved in correction of these errors to keep the frequency of spontaneous mutations very low. We report here a novel mechanism which prevents replicational errors by degrading a potent mutagenic substrate for DNA synthesis. This error-avoiding process is catalysed by a protein encoded by the mutT gene of Escherichia coli, mutations of which increase the occurrence of A.T----C.G transversions 100 to 10,000 times the level of the wild type. Spontaneous oxidation of dGTP forms 8-oxo-7,8-dihydro-2'-dGTP (8-oxodGTP), which is inserted opposite dA and dC residues of template DNA with almost equal efficiency, and the MutT protein specifically degrades 8-oxodGTP to the monophosphate. This indicates that elimination from the nucleotide pool of the oxidized form of guanine nucleotide is important for the high fidelity of DNA synthesis.     

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.     

Fidelity of DNA replication catalysed in vitro on a natural DNA template by the T4 bacteriophage multi-enzyme complex

More than 50 copies of a phi X174 DNA template can be made in 60 min in an in vitro DNA replication system consisting of seven purfied replication proteins isolated from T4 bacteriophage-infected cells. By transfecting with the DNA products and assaying for the reversion of specific amber mutants, the high degree of base-pairing fidelity in this system is revealed; the in vitro system is also shown to respond to the mutagenic effect of Mn2+ and to display strong base-pair context effects on fidelity, as expected from in vivo studies.     

Taq酶体外合成DNA时模板错位机制的探讨

利用Taq DNA聚合酶体外合成DNA过程中,当反应体系中缺少与模板链互补配对的dNTP底物时,产物合成并不会在底物缺失位点处终止,聚合反应继续进行.为研究此复制缺陷现象,设计一系列模板用于DNA体外酶促合成.除了已知的碱基错配机制,笔者发现存在另一种"模板错位"机制,即模板中与底物非Watson-Crick互补配对的碱基位点首先进行收缩滑动,形成模板bulge结构后再继续进行酶促合成反应.这项研究有助于提高DNA样品合成保真度以及继续深入探索体外DNA合成的详细机制.     

Domain of E. coli DNA polymerase I showing sequence homology to T7 DNA polymerase

Escherichia coli contains three DNA polymerases that differ in their size, ability to interact with accessory proteins and biological function. Monomeric DNA polymerase I (Pol I) has a relative molecular mass (Mr) of 103,000 (103K) and is involved primarily in the repair of damaged DNA and the processing of Okazaki fragments; polymerase II is of Mr 120K, and polymerase III has a Mr of 140K, is responsible for the replication of the DNA chromosome and is just one of several proteins that are required for replication. DNA polymerases from bacteriophage as well as those of eukaryotic viral and cellular origin also differ with respect to their size and the number of associated proteins that are required for them to function in replication. However, the template-directed copying of DNA is identical in all cases. The crystal structure of the large proteolytic fragment of Pol I shows that it consists of two domains, the larger of which contains a deep crevice whose dimensions are such that it can bind duplex DNA. The T7 polymerase consists of two subunits, the 80K gene 5 protein and the host-encoded 12K thioredoxin of E. coli. We show here that there is an amino acid sequence homology between at least eight polypeptide segments that form the large cleft in the Klenow fragment and polypeptides in T7 DNA polymerase gene 5 protein, suggesting that this domain evolved from a common precursor. The parts of the Pol I and T7 DNA polymerase molecules that bind the DNA substrate appear to share common structural features, and these features may be shared by all of these varied DNA polymerases.     

Sequential initiation of lagging and leading strand synthesis by two different polymerase complexes at the SV40 DNA replication origin

Enzymatic synthesis of DNA from the simian virus 40 origin of DNA replication has been reconstituted in vitro with eight purified components. DNA polymerase alpha-primase complex first initiates DNA synthesis at the replication origin and continues as the lagging strand polymerase. Subsequently, the DNA polymerase delta complex initiates replication on the leading strand template. Some prokaryotic DNA polymerase complexes can replace the eukaryotic polymerase delta complex. A model for polymerase switching during initiation of DNA replication is presented.     

A model for SOS-lesion-targeted mutations in Escherichia coli

The UmuD'2C protein complex (Escherichia coli pol V) is a low-fidelity DNA polymerase (pol) that copies damaged DNA in the presence of RecA, single-stranded-DNA binding protein (SSB) and the beta,gamma-processivity complex of E. coli pol III (ref. 4). Here we propose a model to explain SOS-lesion-targeted mutagenesis, assigning specific biochemical functions for each protein during translesion synthesis. (SOS lesion-targeted mutagenesis occurs when pol V is induced as part of the SOS response to DNA damage and incorrectly incorporates nucleotides opposite template lesions.) Pol V plus SSB catalyses RecA filament disassembly in the 3' to 5' direction on the template, ahead of the polymerase, in a reaction that does not involve ATP hydrolysis. Concurrent ATP-hydrolysis-driven filament disassembly in the 5' to 3' direction results in a bidirectional stripping of RecA from the template strand. The bidirectional collapse of the RecA filament restricts DNA synthesis by pol V to template sites that are proximal to the lesion, thereby minimizing the occurrence of untargeted mutations at undamaged template sites.     

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.     

Control of spontaneous and damage-induced mutagenesis by SUMO and ubiquitin conjugation

Protein modification by ubiquitin is emerging as a signal for various biological processes in eukaryotes, including regulated proteolysis, but also for non-degradative functions such as protein localization, DNA repair and regulation of chromatin structure. A small ubiquitin-related modifier (SUMO) uses a similar conjugation system that sometimes counteracts the effects of ubiquitination. Ubiquitin and SUMO compete for modification of proliferating cell nuclear antigen (PCNA), an essential processivity factor for DNA replication and repair. Whereas multi-ubiquitination is mediated by components of the RAD6 pathway and promotes error-free repair, SUMO modification is associated with replication. Here we show that RAD6-mediated mono-ubiquitination of PCNA activates translesion DNA synthesis by the damage-tolerant polymerases eta and zeta in yeast. Moreover, polymerase zeta is differentially affected by mono-ubiquitin and SUMO modification of PCNA. Whereas ubiquitination is required for damage-induced mutagenesis, both SUMO and mono-ubiquitin contribute to spontaneous mutagenesis in the absence of DNA damage. Our findings assign a function to SUMO during S phase and demonstrate how ubiquitin and SUMO, by regulating the accuracy of replication and repair, contribute to overall genomic stability.