| 1. |
- Garbacz, Marta A., et al.
(författare)
-
The absence of the catalytic domains of Saccharomyces cerevisiae DNA polymerase ϵ strongly reduces DNA replication fidelity
- 2019
-
Ingår i: Nucleic Acids Research. - : Oxford University Press. - 0305-1048 .- 1362-4962. ; 47:8, s. 3986-3995
-
Tidskriftsartikel (refereegranskat)abstract
- The four B-family DNA polymerases α, δ, ϵ and ζ cooperate to accurately replicate the eukaryotic nuclear genome. Here, we report that a Saccharomyces cerevisiae strain encoding the pol2-16 mutation that lacks Pol ϵ's polymerase and exonuclease activities has increased dNTP concentrations and an increased mutation rate at the CAN1 locus compared to wild type yeast. About half of this mutagenesis disappears upon deleting the REV3 gene encoding the catalytic subunit of Pol ζ. The remaining, still strong, mutator phenotype is synergistically elevated in an msh6Δ strain and has a mutation spectrum characteristic of mistakes made by Pol δ. The results support a model wherein slow-moving replication forks caused by the lack of Pol ϵ's catalytic domains result in greater involvement of mutagenic DNA synthesis by Pol ζ as well as diminished proofreading by Pol δ during replication.
|
|
| 2. |
- Orebaugh, Clinton D, et al.
(författare)
-
Mapping Ribonucleotides Incorporated into DNA by Hydrolytic End-Sequencing.
- 2018
-
Ingår i: Methods in molecular biology (Clifton, N.J.). - New York, NY : Springer New York. - 1940-6029. ; 1672, s. 329-345
-
Tidskriftsartikel (refereegranskat)abstract
- Ribonucleotides embedded within DNA render the DNA sensitive to the formation of single-stranded breaks under alkali conditions. Here, we describe a next-generation sequencing method called hydrolytic end sequencing (HydEn-seq) to map ribonucleotides inserted into the genome of Saccharomyce cerevisiae strains deficient in ribonucleotide excision repair. We use this method to map several genomic features in wild-type and replicase variant yeast strains.
|
|
| 3. |
- Watt, Danielle L., et al.
(författare)
-
Genome-wide analysis of the specificity and mechanisms of replication infidelity driven by imbalanced dNTP pools
- 2016
-
Ingår i: Nucleic Acids Research. - : Oxford University Press (OUP). - 0305-1048 .- 1362-4962. ; 44:4, s. 1669-1680
-
Tidskriftsartikel (övrigt vetenskapligt/konstnärligt)abstract
- The absolute and relative concentrations of the four dNTPs are key determinants of DNA replication fidelity, yet the consequences of altered dNTP pools on replication fidelity have not previously been investigated on a genome-wide scale. Here, we use deep sequencing to determine the types, rates and locations of uncorrected replication errors that accumulate in the nuclear genome of a mismatch repair-deficient diploid yeast strain with elevated dCTP and dTTP concentrations. These imbalanced dNTP pools promote replication errors in specific DNA sequence motifs suggesting increased misinsertion and increased mismatch extension at the expense of proofreading. Interestingly, substitution rates are similar for leading and lagging strand replication, but are higher in regions replicated late in S phase. Remarkably, the rate of single base deletions is preferentially increased in coding sequences and in short rather than long mononucleotides runs. Based on DNA sequence motifs, we propose two distinct mechanisms for generating single base deletions in vivo. Collectively, the results indicate that elevated dCTP and dTTP pools increase mismatch formation and decrease error correction across the nuclear genome, and most strongly increases mutation rates in coding and late replicating sequences.
|
|
| 4. |
- Williams, Jessica S, et al.
(författare)
-
Evidence that processing of ribonucleotides in DNA by topoisomerase 1 is leading-strand specific
- 2015
-
Ingår i: Nature Structural & Molecular Biology. - : Springer Science and Business Media LLC. - 1545-9993 .- 1545-9985. ; 22:4, s. 291-U35
-
Tidskriftsartikel (refereegranskat)abstract
- Ribonucleotides incorporated during DNA replication are removed by RNase H2-dependent ribonucleotide excision repair (RER). In RER-defective yeast, topoisomerase 1 (Top1) incises DNA at unrepaired ribonucleotides, initiating their removal, but this is accompanied by RNA-DNA-damage phenotypes. Here we show that these phenotypes are incurred by a high level of ribonucleotides incorporated by a leading strand-replicase variant, DNA polymerase (Pol) ɛ, but not by orthologous variants of the lagging-strand replicases, Pols α or δ. Moreover, loss of both RNases H1 and H2 is lethal in combination with increased ribonucleotide incorporation by Pol ɛ but not by Pols α or δ. Several explanations for this asymmetry are considered, including the idea that Top1 incision at ribonucleotides relieves torsional stress in the nascent leading strand but not in the nascent lagging strand, in which preexisting nicks prevent the accumulation of superhelical tension.
|
|
| 5. |
- Williams, Jessica S., et al.
(författare)
-
Topoisomerase 1-Mediated Removal of Ribonucleotides from Nascent Leading-Strand DNA
- 2013
-
Ingår i: Molecular Cell. - : Cell Press. - 1097-2765 .- 1097-4164. ; 49:5, s. 1010-1015
-
Tidskriftsartikel (refereegranskat)abstract
- RNase H2-dependent ribonucleotide excision repair (RER) removes ribonucleotides incorporated during DNA replication. When RER is defective, ribonucleotides in the nascent leading strand of the yeast genome are associated with replication stress and genome instability. Here, we provide evidence that topoisomerase 1 (Top1) initiates an independent form of repair to remove ribonucleotides from genomic DNA. This Top1-dependent process activates the S phase checkpoint. Deleting TOP1 reverses this checkpoint activation and also relieves replication stress and genome instability in RER-defective cells. The results reveal an additional removal pathway for a very common lesion in DNA, and they imply that the "dirty" DNA ends created when Top1 incises ribonucleotides in DNA are responsible for the adverse consequences of ribonucleotides in RNase H2-defective cells.
|
|
