Genetic Separation of Sae2 Nuclease Activity from Mre11 Nuclease Functions in Budding Yeast.

Sae2 promotes the repair of DNA double-strand breaks in The role of Sae2 is linked to the Mre11/Rad50/Xrs2 (MRX) complex, which is important for the processing of DNA ends into single-stranded substrates for homologous recombination. Sae2 has intrinsic endonuclease activity, but the role of this activity has not been assessed independently from its functions in promoting Mre11 nuclease activity. Here we identify and characterize separation-of-function mutants that lack intrinsic nuclease activity or the ability to promote Mre11 endonucleolytic activity.

Dna2 is involved in CA strand resection and nascent lagging strand completion at native yeast telomeres.

Post-replicational telomere end processing involves both extension by telomerase and resection to produce 3'-GT-overhangs that extend beyond the complementary 5'-CA-rich strand. Resection must be carefully controlled to maintain telomere length. At short de novo telomeres generated artificially by HO endonuclease in the G2 phase, we show that dna2-defective strains are impaired in both telomere elongation and sequential 5'-CA resection.

Inviability of a DNA2 deletion mutant is due to the DNA damage checkpoint.

Dna2 is a dual polarity exo/endonuclease, and 5' to 3' DNA helicase involved in Okazaki Fragment Processing (OFP) and Double-Strand Break (DSB) Repair. In yeast, DNA2 is an essential gene, as expected for a DNA replication protein. Suppression of the lethality of dna2Δ mutants has been found to occur by two mechanisms: overexpression of RAD27 (scFEN1) , encoding a 5' to 3' exo/endo nuclease that processes Okazaki fragments (OFs) for ligation, or deletion of PIF1, a 5' to 3' helicase involved in mitochondrial recombination, telomerase inhibition and OFP.

Interplay of Mre11 nuclease with Dna2 plus Sgs1 in Rad51-dependent recombinational repair.

The Mre11/Rad50/Xrs2 complex initiates IR repair by binding to the end of a double-strand break, resulting in 5' to 3' exonuclease degradation creating a single-stranded 3' overhang competent for strand invasion into the unbroken chromosome. The nuclease(s) involved are not well understood. Mre11 encodes a nuclease, but it has 3' to 5', rather than 5' to 3' activity.

Mrc1 and DNA polymerase epsilon function together in linking DNA replication and the S phase checkpoint.

Yeast Mrc1, ortholog of metazoan Claspin, is both a central component of normal DNA replication forks and a mediator of the S phase checkpoint. We report that Mrc1 interacts with Pol2, the catalytic subunit of DNA polymerase epsilon, essential for leading-strand DNA replication and for the checkpoint. In unperturbed cells, Mrc1 interacts independently with both the N-terminal and C-terminal halves of Pol2 (Pol2N and Pol2C).

Dpb2p, a noncatalytic subunit of DNA polymerase epsilon, contributes to the fidelity of DNA replication in Saccharomyces cerevisiae.

Most replicases are multi-subunit complexes. DNA polymerase epsilon from Saccharomyces cerevisiae is composed of four subunits: Pol2p, Dpb2p, Dpb3p, and Dpb4p. Pol2p and Dpb2p are essential.

Evidence suggesting that Pif1 helicase functions in DNA replication with the Dna2 helicase/nuclease and DNA polymerase delta.

The precise machineries required for two aspects of eukaryotic DNA replication, Okazaki fragment processing (OFP) and telomere maintenance, are poorly understood. In this work, we present evidence that Saccharomyces cerevisiae Pif1 helicase plays a wider role in DNA replication than previously appreciated and that it likely functions in conjunction with Dna2 helicase/nuclease as a component of the OFP machinery. In addition, we show that Dna2, which is known to associate with telomeres in a cell-cycle-specific manner, may be a new component of the telomere replication apparatus.

A network of multi-tasking proteins at the DNA replication fork preserves genome stability.

To elucidate the network that maintains high fidelity genome replication, we have introduced two conditional mutant alleles of DNA2, an essential DNA replication gene, into each of the approximately 4,700 viable yeast deletion mutants and determined the fitness of the double mutants. Fifty-six DNA2-interacting genes were identified. Clustering analysis of genomic synthetic lethality profiles of each of 43 of the DNA2-interacting genes defines a network (consisting of 322 genes and 876 interactions) whose topology provides clues as to how replication proteins coordinate regulation and repair to protect genome integrity.

Evidence that yeast SGS1, DNA2, SRS2, and FOB1 interact to maintain rDNA stability.

We and others have proposed that faulty processing of arrested replication forks leads to increases in recombination and chromosome instability in Saccharomyces cerevisiae. Now we use the ribosomal DNA locus, which is a good model for all stages of DNA replication, to test this hypothesis. We showed previously that DNA replication pausing at the ribosomal DNA replication fork barrier (RFB) is accompanied by the occurrence of double-strand breaks near the RFB.

Dna2 helicase/nuclease causes replicative fork stalling and double-strand breaks in the ribosomal DNA of Saccharomyces cerevisiae.

We have proposed that faulty processing of arrested replication forks leads to increases in recombination and chromosome instability in Saccharomyces cerevisiae and contributes to the shortened lifespan of dna2 mutants. Now we use the ribosomal DNA locus, which is a good model for all stages of DNA replication, to test this hypothesis. We show directly that DNA replication pausing at the ribosomal DNA replication fork barrier (RFB) is accompanied by the occurrence of double-strand breaks near the RFB.

Dynamic localization of an Okazaki fragment processing protein suggests a novel role in telomere replication.

We have found that the Dna2 helicase-nuclease, thought to be involved in maturation of Okazaki fragments, is a component of telomeric chromatin. We demonstrate a dynamic localization of Dna2p to telomeres that suggests a dual role for Dna2p, one in telomere replication and another, unknown function, perhaps in telomere capping. Both chromatin immunoprecipitation (ChIP) and immunofluorescence show that Dna2p associates with telomeres but not bulk chromosomal DNA in G(1) phase, when there is no telomere replication and the telomere is transcriptionally silenced.

Mutations in DNA replication genes reduce yeast life span.

Surprisingly, the contribution of defects in DNA replication to the determination of yeast life span has never been directly investigated. We show that a replicative yeast helicase/nuclease, encoded by DNA2 and a member of the same helicase subfamily as the RecQ helicases, is required for normal life span. All of the phenotypes of old wild-type cells, for example, extended cell cycle time, age-related transcriptional silencing defects, and nucleolar reorganization, occur after fewer generations in dna2 mutants than in the wild type.