Reconstitution of
            <i>Saccharomyces cerevisiae</i>
            DNA polymerase ε-dependent mismatch repair with purified proteins

Bowen, Nikki; Kolodner, Richard D.

doi:10.1073/pnas.1701753114

Cited by 32 publications

(35 citation statements)

References 57 publications

(105 reference statements)

Supporting

Mentioning

Contrasting

Order By: Relevance

“…The 5′ nick–directed repair reactions contain ATP, Mg 2+ , WT or mutant Msh2–Msh6, replication protein A, Exo1, DNA polymerase δ, PCNA, RFC-Δ1N (RFC-Δ1N is an RFC1–5 complex containing an N-terminal truncation of RFC1 that reduces its DNA binding but leaves all other functions intact ( 59 ); the 3′ nick–directed repair assay reactions in addition contained Mlh1–Pms1. Previous studies have demonstrated that the 5′ nick–directed repair assay depends on Exo1 but does not require Mlh1–Pms1 ( 53 , 58 , 60 ), whereas the 3′ nick–directed repair assay depends on Mlh1–Pms1 ( 53 , 60 ) and under some conditions can be at least partially independent of Exo1 ( 61 ).…”

Section: Resultsmentioning

confidence: 99%

“…In these reactions, Exo1 initiates excision at the 5′ nick in a reaction that is significantly stimulated by Msh2–Msh6 in a mispair recognition–dependent fashion resulting in excision of the mispair ( 58 , 62 ). The resulting single-stranded DNA gap is then filled in by DNA polymerase δ and accessory proteins; in addition, DNA polymerase ϵ can substitute for DNA polymerase δ ( 60 ). At concentrations of ATP (2.5 m m ) that exceed the K d for ATP binding by both the Msh2 and Msh6 nucleotide-binding sites, the Walker A Lys mutant Msh2–Msh6 complexes supported 40–75% of the level of repair supported by WT Msh2–Msh6 ( Fig.…”

Section: Resultsmentioning

confidence: 99%

See 1 more Smart Citation

The properties of Msh2–Msh6 ATP binding mutants suggest a signal amplification mechanism in DNA mismatch repair

Graham¹,

Putnam²,

Kolodner

2018

Journal of Biological Chemistry

Self Cite

View full text Add to dashboard Cite

DNA mismatch repair (MMR) corrects mispaired DNA bases and small insertion/deletion loops generated by DNA replication errors. After binding a mispair, the eukaryotic mispair recognition complex Msh2–Msh6 binds ATP in both of its nucleotide-binding sites, which induces a conformational change resulting in the formation of an Msh2–Msh6 sliding clamp that releases from the mispair and slides freely along the DNA. However, the roles that Msh2–Msh6 sliding clamps play in MMR remain poorly understood. Here, using Saccharomyces cerevisiae, we created Msh2 and Msh6 Walker A nucleotide–binding site mutants that have defects in ATP binding in one or both nucleotide-binding sites of the Msh2–Msh6 heterodimer. We found that these mutations cause a complete MMR defect in vivo. The mutant Msh2–Msh6 complexes exhibited normal mispair recognition and were proficient at recruiting the MMR endonuclease Mlh1–Pms1 to mispaired DNA. At physiological (2.5 mm) ATP concentration, the mutant complexes displayed modest partial defects in supporting MMR in reconstituted Mlh1–Pms1-independent and Mlh1–Pms1-dependent MMR reactions in vitro and in activation of the Mlh1–Pms1 endonuclease and showed a more severe defect at low (0.1 mm) ATP concentration. In contrast, five of the mutants were completely defective and one was mostly defective for sliding clamp formation at high and low ATP concentrations. These findings suggest that mispair-dependent sliding clamp formation triggers binding of additional Msh2–Msh6 complexes and that further recruitment of additional downstream MMR proteins is required for signal amplification of mispair binding during MMR.

show abstract

Section: Resultsmentioning

confidence: 99%

Section: Resultsmentioning

confidence: 99%

The properties of Msh2–Msh6 ATP binding mutants suggest a signal amplification mechanism in DNA mismatch repair

Graham¹,

Putnam²,

Kolodner

2018

Journal of Biological Chemistry

Self Cite

View full text Add to dashboard Cite

show abstract

“…Msh6 interacts with the polymerase processivity clamp PCNA (Flores-Rozas et al 2000), resulting in a fraction of Msh2-Msh6 colocalizing with the replication fork, presumably facilitating the efficient detection of DNA mismatches generated during replication (Hombauer et al 2011). Upon mismatch recognition by MSH heterodimers, MLH heterodimers (primarily Mlh1-Pms1) are recruited to the MSH-mismatch complex, which in turn recruits downstream MMR proteins such as Replication factor C (RFC), PCNA, Exo1, single-strand-binding protein RPA, DNA polymerase d and e, and DNA ligase to promote MMR through excision, resynthesis, and ligation steps [reviewed in Erie (2005, 2015), , and Bowen and Kolodner (2017)].…”

mentioning

confidence: 99%

Genomic Instability Promoted by Overexpression of Mismatch Repair Factors in Yeast: A Model for Understanding Cancer Progression

2018

View full text Add to dashboard Cite

Mismatch repair (MMR) proteins act in spellchecker roles to excise misincorporation errors that occur during DNA replication. Curiously, large-scale analyses of a variety of cancers showed that increased expression of MMR proteins often correlated with tumor aggressiveness, metastasis, and early recurrence. To better understand these observations, we used The Cancer Genome Atlas and Gene Expression across Normal and Tumor tissue databases to analyze MMR protein expression in cancers. We found that the MMR genes and are overexpressed more frequently than , and that and are often cooverexpressed as a result of copy number amplifications of these genes. These observations encouraged us to test the effects of upregulating MMR protein levels in baker's yeast, where we can sensitively monitor genome instability phenotypes associated with cancer initiation and progression. Msh6 overexpression (two- to fourfold) almost completely disrupted mechanisms that prevent recombination between divergent DNA sequences by interacting with the DNA polymerase processivity clamp PCNA and by sequestering the Sgs1 helicase. Importantly, cooverexpression of Msh2 and Msh6 (∼eightfold) conferred, in a PCNA interaction-dependent manner, several genome instability phenotypes including increased mutation rate, increased sensitivity to the DNA replication inhibitor HU and the DNA-damaging agents MMS and 4-nitroquinoline N-oxide, and elevated loss-of-heterozygosity. Msh2 and Msh6 cooverexpression also altered the cell cycle distribution of exponentially growing cells, resulting in an increased fraction of unbudded cells, consistent with a larger percentage of cells in G1. These novel observations suggested that overexpression of MSH factors affected the integrity of the DNA replication fork, causing genome instability phenotypes that could be important for promoting cancer progression.

show abstract

“…Both complexes involved at least two components that were shown in our MS data to interact with CNTD1. Firstly, CNTD1 FH specifically interacts with key regulators of meiotic processes and DNA repair, particularly components of the Replication Factor C (RFC) complex, which in somatic cells acts in the 240 recruitment/activation of the DNA mismatch repair pathway 26,38,39 . CNTD1 FH also interacts with a large number of proteins belonging to the ubiquitylation pathway ( Figure S6b), including components of the SKP1-Cullin-F-Box (SCF; reviewed in 40 ), namely the E2 Conjugating enzyme CDC34 and the novel E3 ubiquitin ligase FBXW9 ( Figure S6b).…”

Section: Rfc and Scf Complexesmentioning

confidence: 99%

“…Replication factor C is a pentameric complex, consisting of RFC1 through RFC5, that loads PCNA during DNA replication 41 . RFC and PCNA have been implicated in activation of MutL endonucleases 26,38,39,42 . Our mass spectrometry data revealed CNTD1 interaction with RFC3 and RFC4 (Supplemental table 1) and was confirmed by anti-HA-immunoprecipitation 250 followed by western blot using anti-RFC3 and RFC4 antibodies ( Figure 4a).…”

Section: Replication Factor C (Rfc) Complexmentioning

confidence: 99%

Cyclin N-Terminal Domain-Containing 1 (CNTD1) coordinates meiotic crossover formation with cell cycle progression in a cyclin-independent manner

Gray

Santiago

Chappie

et al. 2019

Preprint

View full text Add to dashboard Cite

During meiotic prophase I, programmed DNA double-strand breaks repair as non-crossover or crossover events, the latter predominantly occurring via the Class I crossover pathway and requiring the cyclin family member CNTD1. Using an epitope-tagged Cntd1 allele, we show 20 that mouse CNTD1 exists in vivo as a short isoform that lacks the predicted N-terminal cyclin domain and does not bind cyclin-dependent kinases. Instead, we find that CNTD1 associates with Replication Factor C to drive crossover formation and the Skp1-Cullin1-F-Box complex to regulate ubiquitination and subsequent degradation of the WEE1 kinase, thereby indirectly modulating cell cycle progression. We propose that these interactions enable CNTD1 to 25 orchestrate the steps of prophase I and coordinate crossover formation with cellular division.

show abstract

Reconstitution of Saccharomyces cerevisiae DNA polymerase ε-dependent mismatch repair with purified proteins

Cited by 32 publications

References 57 publications

The properties of Msh2–Msh6 ATP binding mutants suggest a signal amplification mechanism in DNA mismatch repair

The properties of Msh2–Msh6 ATP binding mutants suggest a signal amplification mechanism in DNA mismatch repair

Genomic Instability Promoted by Overexpression of Mismatch Repair Factors in Yeast: A Model for Understanding Cancer Progression

Cyclin N-Terminal Domain-Containing 1 (CNTD1) coordinates meiotic crossover formation with cell cycle progression in a cyclin-independent manner

Contact Info

Product

Resources

About