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      Sensitivity to Phosphonoacetic Acid : A New Phenotype to Probe DNA Polymerase δ in Saccharomyces cerevisiae

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      Genetics
      Genetics Society of America

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          Abstract

          A mutant allele (pol3-L612M) of the DNA polymerase delta gene in Saccharomyces cerevisiae that confers sensitivity to the antiviral drug phosphonoacetic acid (PAA) was constructed. We report that PAA-sensitivity tagging DNA polymerases is a useful method for selectively and reversibly inhibiting one type of DNA polymerase. Our initial studies reveal that replication by the L612M-DNA pol delta requires Rad27 flap endonuclease activity since the pol3-L612M strain is not viable in the absence of RAD27 function. The L612M-DNA pol delta also strongly depends on mismatch repair (MMR). Reduced viability is observed in the absence of any of the core MMR proteins-Msh2, Mlh1, or Pms1-and severe sensitivity to PAA is observed in the absence of the core proteins Msh6 or Exo1, but not Msh3. We propose that pol3-L612M cells need the Rad27 flap endonuclease and MMR complexes composed of Msh2/Msh6, Mlh1/Pms1, and Exo1 for correct processing of Okazaki fragments.

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          Most cited references37

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          An Introduction to the Bootstrap

          Statistics is a subject of many uses and surprisingly few effective practitioners. The traditional road to statistical knowledge is blocked, for most, by a formidable wall of mathematics. The approach in An Introduction to the Bootstrap avoids that wall. It arms scientists and engineers, as well as statisticians, with the computational techniques they need to analyze and understand complicated data sets.
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            Three new dominant drug resistance cassettes for gene disruption in Saccharomyces cerevisiae.

            Disruption-deletion cassettes are powerful tools used to study gene function in many organisms, including Saccharomyces cerevisiae. Perhaps the most widely useful of these are the heterologous dominant drug resistance cassettes, which use antibiotic resistance genes from bacteria and fungi as selectable markers. We have created three new dominant drug resistance cassettes by replacing the kanamycin resistance (kan(r)) open reading frame from the kanMX3 and kanMX4 disruption-deletion cassettes (Wach et al., 1994) with open reading frames conferring resistance to the antibiotics hygromycin B (hph), nourseothricin (nat) and bialaphos (pat). The new cassettes, pAG25 (natMX4), pAG29 (patMX4), pAG31 (patMX3), pAG32 (hphMX4), pAG34 (hphMX3) and pAG35 (natMX3), are cloned into pFA6, and so are in all other respects identical to pFA6-kanMX3 and pFA6-kanMX4. Most tools and techniques used with the kanMX plasmids can also be used with the hph, nat and patMX containing plasmids. These new heterologous dominant drug resistance cassettes have unique antibiotic resistance phenotypes and do not affect growth when inserted into the ho locus. These attributes make the cassettes ideally suited for creating S. cerevisiae strains with multiple mutations within a single strain. Copyright 1999 John Wiley & Sons, Ltd.
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              Mismatch repair in replication fidelity, genetic recombination, and cancer biology.

              Mismatch repair stabilizes the cellular genome by correcting DNA replication errors and by blocking recombination events between divergent DNA sequences. The reaction responsible for strand-specific correction of mispaired bases has been highly conserved during evolution, and homologs of bacterial MutS and MutL, which play key roles in mismatch recognition and initiation of repair, have been identified in yeast and mammalian cells. Inactivation of genes encoding these activities results in a large increase in spontaneous mutability, and in the case of mice and men, predisposition to tumor development.
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                Author and article information

                Journal
                Genetics
                Genetics
                Genetics Society of America
                0016-6731
                1943-2631
                June 24 2005
                June 2005
                June 2005
                March 31 2005
                : 170
                : 2
                : 569-580
                Article
                10.1534/genetics.104.040295
                1450396
                15802517
                8b761699-1780-40fb-af35-945173895978
                © 2005
                History

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