A tale of two serines: the effects of histone H2A mutations S122A and S129A on chromosome nondisjunction in Saccharomyces cerevisiae.

Near the C-terminus of histone H2A in the yeast Saccharomyces cerevisiae, there are 2 serines (S122 and S129) that are targets of phosphorylation. The phosphorylation of serine 129 in response to DNA damage is dependent on the Tel1 and Mec1 kinases. In Schizosaccharomyces pombe and S.

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.

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 by whole-genome sequencing of many independent isolates ( = 93) subcloned about 100 times in unstressed growth conditions. The most common alterations were point mutations and small (<100 bp) insertion/deletions ( = 1,337) and mitotic recombination events ( = 1,215).

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.

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

In the yeast , 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 () within this tandem array, and heterozygous for markers flanking the array, we measured interhomolog recombination and intra/sister chromatid exchange in the locus. The rate of intra/sister chromatid recombination exceeded the rate of interhomolog recombination by >10-fold.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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).

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.