Replication of ribonucleotide-containing DNA templates by yeast replicative polymerases

The major replicative DNA polymerases of S. cerevisiae (Pols α, δ, and ?) incorporate substantial numbers of ribonucleotides into DNA during DNA synthesis. When these ribonucleotides are not removed in vivo, they reside in the template strand used for the next round of replication and could potentially reduce replication efficiency and fidelity. To examine if the presence of ribonucleotides in a DNA template impede DNA synthesis, we determined the efficiency with which Pols α, δ, and ? copy DNA templates containing a single ribonucleotide. All three polymerases can replicate past ribonucleotides. Relative to all-DNA templates, bypass of ribo-containing templates is slightly reduced, to extents that depend on the identity of the ribo and the sequence context in which it resides. Bypass efficiencies for Pols δ and ? were increased by increasing the dNTP concentrations to those induced by cellular stress, and in the case of Pol ?, by inactivating the 3'-exonuclease activity. Overall, ribonucleotide bypass efficiencies are comparable to, and usually exceed, those for the common oxidative stress-induced lesion 8-oxo-guanine.     

DNA lesion bypass polymerases open up

Structures of catalytic fragments of two DNA lesion bypass DNA polymerases, yeast DNA polymerase eta and an archeon DinB homolog, have recently been solved. These structures share several common architectural and structural features observed in other DNA polymerases, including a hand-like architecture with fingers, palm, and thumb subdomains. The new structures provide the first structural insights into DNA lesion bypass. The fingers and thumb are smaller than those in other DNA polymerases. Modeled substrates suggest that the fingers in the vicinity of the incoming nucleotide is closed, a conformation not previously observed for an unliganded polymerase. However, the template binding pocket appears to be more open, indicating that for DNA polymerase eta, a covalently linked thymine-thymine dimer could be accommodated.     

Accuracy, lesion bypass, strand displacement and translocation by DNA polymerases

The structures of DNA polymerases from different families show common features and significant differences that shed light on the ability of these enzymes to accurately copy DNA and translocate. The structure of a B family DNA polymerase from phage RB69 exhibits an active-site closing conformational change in the fingers domain upon forming a ternary complex with primer template in deoxynucleoside triphosphate. The rotation of the fingers domain alpha-helices by 60 degrees upon dNTP binding is analogous to the changes seen in other families of polymerases. When the 3' terminus is bound to the editing 3' exonuclease active site, the orientation of the DNA helix axis changes by 40 degrees and the thumb domain re-orients with the DNA. Structures of substrate and product complexes of T7 RNA polymerase, a structural homologue of T7 DNA polymerase, show that family polymerases use the rotation conformational change of the fingers domain to translocate down the DNA. The fingers opening rotation that results in translocation is powered by the release of the product pyrophosphate and also enables the Pol I family polymerases to function as a helicase in displacing the downstream non-template strand from the template strand.     

Specificity of DNA lesion bypass by the yeast DNA polymerase eta

DNA polymerase eta (Pol(eta), xeroderma pigmentosum variant, or Rad30) plays an important role in an error-free response to unrepaired UV damage during replication. It faithfully synthesizes DNA opposite a thymine-thymine cis-syn-cyclobutane dimer. We have purified the yeast Pol(eta) and studied its lesion bypass activity in vitro with various types of DNA damage. The yeast Pol(eta) lacked a nuclease or a proofreading activity. It efficiently bypassed 8-oxoguanine, incorporating C, A, and G opposite the lesion with a relative efficiency of approximately 100:56:14, respectively. The yeast Pol(eta) efficiently incorporated a C opposite an acetylaminofluorene-modified G, and efficiently inserted a G or less frequently an A opposite an apurinic/apyrimidinic (AP) site but was unable to extend the DNA synthesis further in both cases. However, some continued DNA synthesis was observed in the presence of the yeast Pol(zeta) following the Pol(eta) action opposite an AP site, achieving true lesion bypass. In contrast, the yeast Pol(alpha) was able to bypass efficiently a template AP site, predominantly incorporating an A residue opposite the lesion. These results suggest that other than UV damage, Pol(eta) may also play a role in bypassing additional DNA lesions, some of which can be error-prone.     

Defining the position of the switches between replicative and bypass DNA polymerases

Cells contain specialized DNA polymerases that are able to copy past lesions with an associated risk of generating mutations, the major cause of cancer. Here, we reconstitute translesion synthesis (TLS) using the replicative (Pol III) and major bypass (Pol V) DNA polymerases from Escherichia coli in the presence of accessory factors. When the replicative polymerase disconnects from the template in the vicinity of a lesion, Pol V binds the blocked replication intermediate and forms a stable complex by means of a dual interaction with the tip of the RecA filament and the beta-clamp, the processivity factor donated by the blocked Pol III holoenzyme. Both interactions are required to confer to Pol V the processivity that will allow it synthesize, in a single binding event, a TLS patch long enough to support further extension by Pol III. In the absence of these accessory factors, the patch synthesized by Pol V is too short, being degraded by the Pol III-associated exonuclease activity that senses the distortion induced by the lesion, thus leading to an aborted bypass process.     

Mechanism of strand displacement synthesis by DNA replicative polymerases

Replicative holoenzymes exhibit rapid and processive primer extension DNA synthesis, but inefficient strand displacement DNA synthesis. We investigated the bacteriophage T4 and T7 holoenzymes primer extension activity and strand displacement activity on a DNA hairpin substrate manipulated by a magnetic trap. Holoenzyme primer extension activity is moderately hindered by the applied force. In contrast, the strand displacement activity is strongly stimulated by the applied force; DNA polymerization is favoured at high force, while a processive exonuclease activity is triggered at low force. We propose that the DNA fork upstream of the holoenzyme generates a regression pressure which inhibits the polymerization-driven forward motion of the holoenzyme. The inhibition is generated by the distortion of the template strand within the polymerization active site thereby shifting the equilibrium to a DNA-protein exonuclease conformation. We conclude that stalling of the holoenzyme induced by the fork regression pressure is the basis for the inefficient strand displacement synthesis characteristic of replicative polymerases. The resulting processive exonuclease activity may be relevant in replisome disassembly to reset a stalled replication fork to a symmetrical situation. Our findings offer interesting applications for single-molecule DNA sequencing.     

Evidence for interplay among yeast replicative DNA polymerases alpha, delta and epsilon from studies of exonuclease and polymerase active site mutations

Background  

DNA polymerase ε (Pol ε) is essential for S-phase replication, DNA damage repair and checkpoint control in yeast. A pol2-Y831A mutation leading to a tyrosine to alanine change in the Pol ε active site does not cause growth defects and confers a mutator phenotype that is normally subtle but strong in a mismatch repair-deficient strain. Here we investigate the mechanism responsible for the mutator effect.     

A euryarchaeal histone modulates strand displacement synthesis by replicative DNA polymerases

Euryarchaeota and Crenarchaeota, the two main lineages of the domain Archaea, encode different chromatin proteins and differ in the use of replicative DNA polymerases. Crenarchaea possess a single family B DNA polymerase (PolB), which is capable of strand displacement modulated by the chromatin proteins Cren7 and Sul7d. Euryarchaea have two distinct replicative DNA polymerases, PolB and PolD, a family D DNA polymerase. Here we characterized the strand displacement activities of PolB and PolD from the hyperthermophilic euryarchaeon Pyrococcus furiosus and investigated the influence of HPfA1, a homolog of eukaryotic histones from P. furiosus, on these activities. We showed that both PolB and PolD were efficient in strand displacement. HPfA1 inhibited DNA strand displacement by both DNA polymerases but exhibited little effect on the displacement of a RNA strand annealed to single-stranded template DNA. This is consistent with the finding that HPfA1 bound more tightly to double-stranded DNA than to a RNA:DNA hybrid. Our results suggest that, although crenarchaea and euryarchaea differ in chromosomal packaging, they share similar mechanisms in modulating strand displacement by DNA polymerases during lagging strand DNA synthesis.     

Fidelity of mammalian DNA replication and replicative DNA polymerases.

Current models suggest that two or more DNA polymerases may be required for high-fidelity semiconservative DNA replication in eukaryotic cells. In the present study, we directly compare the fidelity of SV40 origin-dependent DNA replication in human cell extracts to the fidelity of mammalian DNA polymerases alpha, delta, and epsilon using lacZ alpha of M13mp2 as a reporter gene. Their fidelity, in decreasing order, is replication greater than or equal to pol epsilon greater than pol delta greater than pol alpha. DNA sequence analysis of mutants derived from extract reactions suggests that replication is accurate when considering single-base substitutions, single-base frameshifts, and larger deletions. The exonuclease-containing calf thymus DNA polymerase epsilon is also highly accurate. When high concentrations of deoxynucleoside triphosphates and deoxyguanosine monophosphate are included in the pol epsilon reaction, both base substitution and frameshift error rates increase. This response suggests that exonucleolytic proofreading contributes to the high base substitution and frameshift fidelity. Exonuclease-containing calf thymus DNA polymerase delta, which requires proliferating cell nuclear antigen for efficient synthesis, is significantly less accurate than pol epsilon. In contrast to pol epsilon, pol delta generates errors during synthesis at a relatively modest concentration of deoxynucleoside triphosphates (100 microM), and the error rate did not increase upon addition of adenosine monophosphate. Thus, we are as yet unable to demonstrate that exonucleolytic proofreading contributes to accuracy during synthesis by DNA polymerase delta. The four-subunit DNA polymerase alpha-primase complex from both HeLa cells and calf thymus is the least accurate replicative polymerase. Fidelity is similar whether the enzyme is assayed immediately after purification or after being stored frozen.(ABSTRACT TRUNCATED AT 250 WORDS)     

Use of yeast transformation by oligonucleotides to study DNA lesion bypass in vivo

We have studied mutagenic specificities of DNA lesions in vivo in yeast CYC1 oligonucleotide transformation assay. We introduced two lesions into oligonucleotides. One was a nucleoside analog, 3,4-dihydro-6H,8H-pyrimido[4,5-c][1,2]oxazin-7-one 2'-deoxyriboside (dP), which is highly mutagenic to bacteria. It is supposed to be a miscoding, but otherwise good template for DNA polymerases. The other lesion was the TT pyrimidine(6-4)pyrimidone photoproduct, one of the typical UV lesions, which blocks DNA replication. These oligonucleotides were used to transform yeast cyc1 mutants with ochre nonsense mutation to Cyc1+. As expected from its templating properties in vitro, the transforming activity of dP-containing oligonucleotides was similar to those of unmodified oligonucleotides. Results indicated that dP may direct incorporation of guanine and adenine at a ratio of 1:20 or more in vivo. An oligonucleotide containing the photoproduct showed the transforming activity of as low as 3-5% of that of the corresponding unmodified oligonucleotide. This bypass absolutely required REV1 gene. The sequence analysis of the transformants has shown that the lesion was read as TT and TC at a ratio of 3:7, indicating its high mutagenic potential.     

Evidence for template-specific sites in DNA polymerases

Using rabbit hemoglobin messenger RNA as template, $\text{E. coli}$ polymerase I produces poly (dT), poly (dA)·(dT) and antimessenger DNA products. Mild heating of the enzyme causes a differential loss in activity as indicated by three rates of inactivation for the three types of synthesis. Heat inactivation studies have also been carried out with DNA polymerases from oncogenic RNA viruses and mammalian sources using various homopolymer-oligomer pairs as primertemplates. In general, for any given enzyme these synthetic primer-templates reveal different extents of inactivation of the polymerase. These findings may be interpreted to suggest a) that the binding of DNA polymerase to various primer-templates produces conformational changes in the enzyme which are dependent on the type of template bound, or b) that many, if not all, DNA polymerases have different subsites for different templates.     

The fidelity of DNA synthesis by eukaryotic replicative and translesion synthesis polymerases

In their seminal publication describing the structure of the DNA double helix , Watson and Crick wrote what may be one of the greatest understatements in the scientific literature, namely that ＂It has not escaped our notice that the specific pairing we have postulated immediately suggests a possible copying mechanism for the genetic material.＂ Half a century later, we more fully appreciate what a huge challenge it is to replicate six billion nucleotides with the accuracy needed to stably maintain the human genome over many generations. This challenge is perhaps greater than was realized 50 years ago, because subsequent studies have revealed that the genome can be destabilized not only by environmental stresses that generate a large number and variety of potentially cytotoxic and mutagenic lesions in DNA but also by various sequence motifs of normal DNA that present challenges to replication. Towards a better understanding of the many determinants of genome stability, this chapter reviews the fidelity with which undamaged and damaged DNA is copied, with a focus on the eukaryotic B- and Y-family DNA polymerases, and considers how this fidelity is achieved.     

DNA polymerases from bakers' yeast.

Two DNA polymerases are present in extracts of commercial bakers' yeast and wild type Saccharomyces cerevisiae grown aerobically to late log phase. Yeast DNA polymerase I and yeast DNA polymerase II can be separated by DEAE-cellulose, hydroxylapatite, and denatured DNA-cellulose chromatography from the postmitochondrial supernatants of yeast lysates. The yeast polymerases are both of high molecular weight (greater than 100,000) but are clearly separate species by the lack of immunological cross-reactivity. Analysis of associated enzyme activities and other reaction properties of yeast DNA polymerases provides additional evidence for distinguishing the two species. Enzyme I has no associated nuclease activity but does carry out pyrophosphate exchange and pyrophosphorolysis reactions, and has an associated 3'-exonuclease activity. Enzyme I does not degrade deoxynucleoside triphosphates and cannot utilize a mismatched template. Enzyme II does carry out a template-dependent deoxynucleoside triphosphate degradation reaction and can excise mismatched 3'-nucleotides from suitable template systems. Earlier studies have shown that both Enzyme I and Enzyme II are inhibited by N-ethylmaleimide. The yeast enzymes are not identical to any known eukaryotic or prokaryotic DNA polymerases. In general, Enzyme I appears to be most similar to eukaryotic DNA polymerase alpha and Ezyme II exhibits properties of prokaryotic DNA polymerases II and III.     

Quantitative analysis of the efficiency and mutagenic spectra of abasic lesion bypass catalyzed by human Y-family DNA polymerases

Higher eukaryotes encode various Y-family DNA polymerases to perform global DNA lesion bypass. To provide complete mutation spectra for abasic lesion bypass, we employed short oligonucleotide sequencing assays to determine the sequences of abasic lesion bypass products synthesized by human Y-family DNA polymerases eta (hPolη), iota (hPolι) and kappa (hPolκ). The fourth human Y-family DNA polymerase, Rev1, failed to generate full-length lesion bypass products after 3 h. The results indicate that hPolι generates mutations with a frequency from 10 to 80% during each nucleotide incorporation event. In contrast, hPolη is the least error prone, generating the fewest mutations in the vicinity of the abasic lesion and inserting dAMP with a frequency of 67% opposite the abasic site. While the error frequency of hPolκ is intermediate to those of hPolη and hPolι, hPolκ has the highest potential to create frameshift mutations opposite the abasic site. Moreover, the time (t₅₀^bypass) required to bypass 50% of the abasic lesions encountered by hPolη, hPolι and hPolκ was 4.6, 112 and 1 823 s, respectively. These t₅₀^bypass values indicate that, among the enzymes, hPolη has the highest abasic lesion bypass efficiency. Together, our data suggest that hPolη is best suited to perform abasic lesion bypass in vivo.     

Structure-dependent bypass of DNA interstrand crosslinks by translesion synthesis polymerases

DNA interstrand crosslinks (ICLs), inhibit DNA metabolism by covalently linking two strands of DNA and are formed by antitumor agents such as cisplatin and nitrogen mustards. Multiple complex repair pathways of ICLs exist in humans that share translesion synthesis (TLS) past a partially processed ICL as a common step. We have generated site-specific major groove ICLs and studied the ability of Y-family polymerases and Pol ζ to bypass ICLs that induce different degrees of distortion in DNA. Two main factors influenced the efficiency of ICL bypass: the length of the dsDNA flanking the ICL and the length of the crosslink bridging two bases. Our study shows that ICLs can readily be bypassed by TLS polymerases if they are appropriately processed and that the structure of the ICL influences which polymerases are able to read through it.     

Human DNA polymerase epsilon is expressed during cell proliferation in a manner characteristic of replicative DNA polymerases.

In order to shed light on the role of mammalian DNA polymerase epsilon we studied the expression of mRNA for the human enzyme during cell proliferation and during the cell cycle. Steady-state levels of mRNA encoding DNA polymerase epsilon were elevated dramatically when quiescent (G0) cells were stimulated to proliferate (G1/S) in a similar manner to those of DNA polymerase alpha. Message levels of DNA polymerase beta were unchanged in similar experiments. The concentration of immunoreactive DNA polymerase epsilon was also much higher in extracts from proliferating tissues than in those from non-proliferating or slowly proliferating tissues. The level of DNA polymerase epsilon mRNA in actively cycling cells synchronized with nocodazole and in cells fractionated by counterflow centrifugal elutriation showed weaker variation, being at its highest at the G1/S stage boundary. The results presented strongly suggest that mammalian DNA polymerase epsilon is involved in the replication of chromosomal DNA and/or in a repair process that may be substantially activated during the replication of chromosomal DNA. A hypothetical role for DNA polymerase epsilon in a repair process coupled to replication is discussed.     

Error-prone lesion bypass by human DNA polymerase eta

DNA lesion bypass is an important cellular response to genomic damage during replication. Human DNA polymerase η (Polη), encoded by the Xeroderma pigmentosum variant (XPV) gene, is known for its activity of error-free translesion synthesis opposite a TT cis-syn cyclobutane dimer. Using purified human Polη, we have examined bypass activities of this polymerase opposite several other DNA lesions. Human Polη efficiently bypassed a template 8-oxoguanine, incorporating an A or a C opposite the lesion with similar efficiencies. Human Polη effectively bypassed a template abasic site, incorporating an A and less frequently a G opposite the lesion. Significant –1 deletion was also observed when the template base 5′ to the abasic site is a T. Human Polη partially bypassed a template (+)-trans-anti-benzo[a]pyrene-N²-dG and predominantly incorporated an A, less frequently a T, and least frequently a G or a C opposite the lesion. This specificity of nucleotide incorporation correlates well with the known mutation spectrum of (+)-trans-anti-benzo[a]pyrene-N²-dG lesion in mammalian cells. These results show that human Polη is capable of error-prone translesion DNA syntheses in vitro and suggest that Polη may bypass certain lesions with a mutagenic consequence in humans.