Global genomic instability caused by reduced expression of DNA polymerase ε in yeast.

SignificanceAlthough most studies of the genetic regulation of genome stability involve an analysis of mutations within the coding sequences of genes required for DNA replication or DNA repair, recent studies in yeast show that reduced levels of wild-type enzymes can also produce a mutator phenotype. By whole-genome sequencing and other methods, we find that reduced levels of the wild-type DNA polymerase ε in yeast greatly increase the rates of mitotic recombination, aneuploidy, and single-base mutations. The observed pattern of genome instability is different from those observed in yeast strains with reduced levels of the other replicative DNA polymerases, Pol α and Pol δ. These observations are relevant to our understanding of cancer and other diseases associated with genetic instability.

Genome-wide mapping of spontaneous genetic alterations in diploid yeast cells.

Genomic alterations including single-base mutations, deletions and duplications, translocations, mitotic recombination events, and chromosome aneuploidy generate genetic diversity. We examined the rates of all of these genetic changes in a diploid strain of Saccharomyces cerevisiae by whole-genome sequencing of many independent isolates (n = 93) subcloned about 100 times in unstressed growth conditions. The most common alterations were point mutations and small (<100 bp) insertion/deletions (n = 1,337) and mitotic recombination events (n = 1,215). The diploid cells of most eukaryotes are heterozygous for many single-nucleotide polymorphisms (SNPs). During mitotic cell divisions, recombination can produce derivatives of these cells that have become homozygous for the polymorphisms, termed loss-of-heterozygosity (LOH) events. LOH events can change the phenotype of the cells and contribute to tumor formation in humans. We observed two types of LOH events: interstitial events (conversions) resulting in a short LOH tract (usually less than 15 kb) and terminal events (mostly cross-overs) in which the LOH tract extends to the end of the chromosome. These two types of LOH events had different distributions, suggesting that they may have initiated by different mechanisms. Based on our results, we present a method of calculating the probability of an LOH event for individual SNPs located throughout the genome. We also identified several hotspots for chromosomal rearrangements (large deletions and duplications). Our results provide insights into the relative importance of different types of genetic alterations produced during vegetative growth.

Meeting report of the third annual Tri-Service Microbiome Consortium symposium.

The Tri-Service Microbiome Consortium (TSMC) was founded to enhance collaboration, coordination, and communication of microbiome research among U.S. Department of Defense (DoD) organizations and to facilitate resource, material and information sharing among consortium members. The 2019 annual symposium was held 22-24 October 2019 at Wright-Patterson Air Force Base in Dayton, OH. Presentations and discussions centered on microbiome-related topics within five broad thematic areas: 1) human microbiomes; 2) transitioning products into Warfighter solutions; 3) environmental microbiomes; 4) engineering microbiomes; and 5) microbiome simulation and characterization. Collectively, the symposium provided an update on the scope of current DoD microbiome research efforts, highlighted innovative research being done in academia and industry that can be leveraged by the DoD, and fostered collaborative opportunities. This report summarizes the presentations and outcomes of the 3rd annual TSMC symposium.

Properties of Mitotic and Meiotic Recombination in the Tandemly-Repeated CUP1 Gene Cluster in the Yeast Saccharomyces cerevisiae.

In the yeast Saccharomyces cerevisiae, the genes encoding the metallothionein protein Cup1 are located in a tandem array on chromosome VIII. Using a diploid strain that is heterozygous for an insertion of a selectable marker (URA3) within this tandem array, and heterozygous for markers flanking the array, we measured interhomolog recombination and intra/sister chromatid exchange in the CUP1 locus. The rate of intra/sister chromatid recombination exceeded the rate of interhomolog recombination by >10-fold. Loss of the Rad51 and Rad52 proteins, required for most interhomolog recombination, led to a relatively small reduction of recombination in the CUP1 array. Although interhomolog mitotic recombination in the CUP1 locus is elevated relative to the average genomic region, we found that interhomolog meiotic recombination in the array is reduced compared to most regions. Lastly, we showed that high levels of copper (previously shown to elevate CUP1 transcription) lead to a substantial elevation in rate of both interhomolog and intra/sister chromatid recombination in the CUP1 array; recombination events that delete the URA3 insertion from the CUP1 array occur at a rate of >10-3/division in unselected cells. This rate is almost three orders of magnitude higher than observed for mitotic recombination events involving single-copy genes. In summary, our study shows that some of the basic properties of recombination differ considerably between single-copy and tandemly-repeated genes.

Structures of naturally evolved CUP1 tandem arrays in yeast indicate that these arrays are generated by unequal nonhomologous recombination.

An important issue in genome evolution is the mechanism by which tandem duplications are generated from single-copy genes. In the yeast Saccharomyces cerevisiae, most strains contain tandemly duplicated copies of CUP1, a gene that encodes a copper-binding metallothionein. By screening 101 natural isolates of S. cerevisiae, we identified five different types of CUP1-containing repeats, as well as strains that only had one copy of CUP1. A comparison of the DNA sequences of these strains indicates that the CUP1 tandem arrays were generated by unequal nonhomologous recombination events from strains that had one CUP1 gene.

The efficiency and fidelity of 8-oxo-guanine bypass by DNA polymerases delta and eta.

A DNA lesion created by oxidative stress is 7,8-dihydro-8-oxo-guanine (8-oxoG). Because 8-oxoG can mispair with adenine during DNA synthesis, it is of interest to understand the efficiency and fidelity of 8-oxoG bypass by DNA polymerases. We quantify bypass parameters for two DNA polymerases implicated in 8-oxoG bypass, Pols delta and eta. Yeast Pol delta and yeast Pol eta both bypass 8-oxoG and misincorporate adenine during bypass. However, yeast Pol eta is 10-fold more efficient than Pol delta, and following bypass Pol eta switches to less processive synthesis, similar to that observed during bypass of a cis-syn thymine-thymine dimer. Moreover, yeast Pol eta is at least 10-fold more accurate than yeast Pol delta during 8-oxoG bypass. These differences are maintained in the presence of the accessory proteins RFC, PCNA and RPA and are consistent with the established role of Pol eta in suppressing ogg1-dependent mutagenesis in yeast. Surprisingly different results are obtained with human and mouse Pol eta. Both mammalian enzymes bypass 8-oxoG efficiently, but they do so less processively, without a switch point and with much lower fidelity than yeast Pol eta. The fact that yeast and mammalian Pol eta have intrinsically different catalytic properties has potential biological implications.

Reduced levels of DNA polymerase delta induce chromosome fragile site instability in yeast.

Specific regions of genomes (fragile sites) are hot spots for the chromosome rearrangements that are associated with many types of cancer cells. Understanding the molecular mechanisms regulating the stability of chromosome fragile sites, therefore, has important implications in cancer biology. We previously identified two chromosome fragile sites in Saccharomyces cerevisiae that were induced in response to the reduced expression of Pol1p, the catalytic subunit of DNA polymerase alpha. In the study presented here, we show that reduced levels of Pol3p, the catalytic subunit of DNA polymerase delta, induce instability at these same sites and lead to the generation of a variety of chromosomal aberrations. These findings demonstrate that a change in the stoichiometry of replicative DNA polymerases results in recombinogenic DNA lesions, presumably double-strand DNA breaks.

In-frame overlapping genes: the challenges for regulating gene expression.

In-frame overlapping genes in phage, plasmid and bacterial genomes permit synthesis of more than one form of protein from the same gene. Having one gene entirely within another rather than two separate genes presumably precludes recombination events between the identical sequences. However, studies of such gene pairs indicate that the overlapping arrangement can make regulation of the genes more difficult. Here, we extend studies of in-frame overlapping genes II and X from filamentous phage f1 to determine if translational controls are required to regulate the gene properly. These genes encode proteins (pII and pX) with essential but opposing roles in phage DNA replication. They must be tightly regulated to maintain production of the proteins at relative steady state levels that permit continuous replication without killing the host. To determine why little or no pX appears to be made on the gene II/X mRNA, gene II translation was lowered by progressively deleting into the gene II initiator region. Increased pX translation resulted, suggesting that elongating ribosomes on the gene II mRNA interfere with internal initiation on the gene X ribosome binding site and limit gene X translation. As judged from systematically lowering the efficiency of suppression at a gene II amber codon upstream from the gene X start, the already modest level of gene II translation would have to be reduced by more than twofold to relieve all interference with internal initiation. Further downregulation of gene X expression proved to be required to maintain pX at levels relative to pII that are tolerated by the cell. Site-directed mutagenesis and nuclease mapping revealed that the gene X initiation site is sequestered in an extended RNA secondary structure that lowers gene X translation on the two mRNAs encoding it. The more general implications of the results for expression of in-frame overlapping genes are discussed.

A lysine residue in the fingers subdomain of T7 DNA polymerase modulates the miscoding potential of 8-oxo-7,8-dihydroguanosine.

8-oxo-7,8-dihydroguanosine (8oG) is a highly mutagenic DNA lesion that stably pairs with adenosine, forming 8oG(syn).dA(anti) Hoogsteen base pairs. DNA polymerases show different propensities to insert dCMP or dAMP opposite 8oG, but the molecular mechanisms that determine faithful or mutagenic bypass are poorly understood. Here, we report kinetic and structural data providing evidence that, in T7 DNA polymerase, residue Lys536 is responsible for attenuating the miscoding potential of 8oG. The Lys536Ala polymerase shows a significant increase in mutagenic 8oG bypass versus wild-type polymerase, and a crystal structure of the Lys536Ala mutant reveals a closed complex with an 8oG(syn).dATP mismatch in the polymerase active site, in contrast to the unproductive, open complex previously obtained by using wild-type polymerase. We propose that Lys536 acts as a steric and/or electrostatic filter that attenuates the miscoding potential of 8oG by normally interfering with the binding of 8oG in a syn conformation that pairs with dATP.

Structural basis for the dual coding potential of 8-oxoguanosine by a high-fidelity DNA polymerase.

Accurate DNA replication involves polymerases with high nucleotide selectivity and proofreading activity. We show here why both fidelity mechanisms fail when normally accurate T7 DNA polymerase bypasses the common oxidative lesion 8-oxo-7, 8-dihydro-2'-deoxyguanosine (8oG). The crystal structure of the polymerase with 8oG templating dC insertion shows that the O8 oxygen is tolerated by strong kinking of the DNA template. A model of a corresponding structure with dATP predicts steric and electrostatic clashes that would reduce but not eliminate insertion of dA. The structure of a postinsertional complex shows 8oG(syn).dA (anti) in a Hoogsteen-like base pair at the 3' terminus, and polymerase interactions with the minor groove surface of the mismatch that mimic those with undamaged, matched base pairs. This explains why translesion synthesis is permitted without proofreading of an 8oG.dA mismatch, thus providing insight into the high mutagenic potential of 8oG.

Enzymatic switching for efficient and accurate translesion DNA replication.

When cyclobutane pyrimidine dimers stall DNA replication by DNA polymerase (Pol) delta or epsilon, a switch occurs to allow translesion synthesis by DNA polymerase eta, followed by another switch that allows normal replication to resume. In the present study, we investigate these switches using Saccharomyces cerevisiae Pol delta, Pol epsilon and Pol eta and a series of matched and mismatched primer templates that mimic each incorporation needed to completely bypass a cis-syn thymine-thymine (TT) dimer. We report a complementary pattern of substrate use indicating that enzymatic switching involving localized translesion synthesis by Pol eta and mismatch excision and polymerization by a major replicative polymerase can account for the efficient and accurate dimer bypass known to suppress sunlight-induced mutagenesis and skin cancer.

Efficiency, fidelity and enzymatic switching during translesion DNA synthesis.

More than half of the 16 human DNA polymerases may have some role in DNA replication and potentially modulate the biological effects of DNA template lesions that impede replication fork progression. As one approach to understand how multiple polymerases are coordinated at the fork, we recently quantified the efficiency and fidelity with which one particular translesion synthesis enzyme, human DNA polymerase eta, copies templates containing cis-syn thymine dimers. Several observations from that study were unanticipated. Here we discuss the structural and biological implications of those results in light of earlier studies of translesion synthesis.

Investigating the role of the little finger domain of Y-family DNA polymerases in low fidelity synthesis and translesion replication.

Dpo4 and Dbh are Y-family polymerases that originate from two closely related strains of Sulfolobaceae. Quite surprisingly, however, the two polymerases exhibit different enzymatic properties in vitro. For example, Dpo4 can replicate past a variety of DNA lesions, yet Dbh does so with a much lower efficiency. When replicating undamaged DNA, Dpo4 is prone to make base pair substitutions, whereas Dbh predominantly makes single-base deletions. Overall, the two proteins are 54% identical, but the greatest divergence is found in their respective little finger (LF) domains, which are only 41% identical. To investigate the role of the LF domain in the fidelity and lesion-bypassing abilities of Y-family polymerases, we have generated chimeras of Dpo4 and Dbh in which their LF domains have been interchanged. Interestingly, by replacing the LF domain of Dbh with that of Dpo4, the enzymatic properties of the chimeric enzyme are more Dpo4-like in that the enzyme is more processive, can bypass an abasic site and a thymine-thymine cyclobutane pyrimidine dimer, and predominantly makes base pair substitutions when replicating undamaged DNA. The converse is true for the Dpo4-LF-Dbh chimera, which is more Dbh-like in its processivity and ability to bypass DNA adducts and generate single-base deletion errors. Our studies indicate that the unique but variable LF domain of Y-family polymerases plays a major role in determining the enzymatic and biological properties of each individual Y-family member.

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.

The efficiency and specificity of apurinic/apyrimidinic site bypass by human DNA polymerase eta and Sulfolobus solfataricus Dpo4.

One of the most common DNA lesions arising in cells is an apurinic/apyrimidinic (AP) site resulting from base loss. Although a template strand AP site impedes DNA synthesis, translesion synthesis (TLS) DNA polymerases can bypass an AP site. Because this bypass is expected to be highly mutagenic because of loss of base coding potential, here we quantify the efficiency and the specificity of AP site bypass by two Y family TLS enzymes, Sulfolobus solfataricus DNA polymerase 4 (Dpo4) and human DNA polymerase eta (Pol eta). During a single cycle of processive DNA synthesis, Dpo4 and Pol eta bypass synthetic AP sites with 13-30 and 10-13%, respectively, of the bypass efficiency for undamaged bases in the same sequence contexts. These efficiencies are higher than for the A family, exonuclease-deficient Klenow fragment of Escherichia coli DNA polymerase I. We then determined AP site bypass specificity for complete bypass, requiring insertion or misalignment at the AP site followed by multiple incorporations using the aberrant primer templates. Although Dpo4, Pol eta, and Klenow polymerase have different fidelity when copying undamaged DNA, bypass of AP sites lacking A or G by all three polymerases is nearly 100% mutagenic. The majority (70-80%) of bypass events made by all three polymerases are insertion of dAMP opposite the AP site. Single base deletion errors comprise 10-25% of bypass events, with other base insertions observed at lower rates. Given that mammalian cells contain five polymerases implicated in TLS, and given that a large number of AP sites are generated per mammalian cell per day, even moderately efficient AP site bypass could be a source of substitution and frameshift mutagenesis in vivo.

Low fidelity DNA synthesis by a y family DNA polymerase due to misalignment in the active site.

Sulfolobus solfataricus DNA polymerase IV (Dpo4) is a member of the Y family of DNA polymerases whose crystal structure has recently been solved. As a model for other evolutionarily conserved Y family members that perform translesion DNA synthesis and have low fidelity, we describe here the base substitution and frameshift fidelity of DNA synthesis by Dpo4. Dpo4 generates all 12 base-base mismatches at high rates, 11 of which are similar to those of its human homolog, DNA polymerase kappa. This result is consistent with the Dpo4 structure, implying lower geometric selection for correct base pairs. Surprisingly, Dpo4 generates C.dCMP mismatches at an unusually high average rate and preferentially at cytosine flanked by 5'-template guanine. Dpo4 also has very low frameshift fidelity and frequently generates deletions of even noniterated nucleotides, especially cytosine flanked by a 5'-template guanine. Both unusual features of error specificity suggest that Dpo4 can incorporate dNTP precursors when two template nucleotides are present in the active site binding pocket. These results have implications for mutagenesis resulting from DNA synthesis by Y family polymerases.