Abstract
To investigate nuclear DNA replication enzymology in vivo, we have studied Saccharomyces cerevisiae strains containing a pol2-16 mutation that inactivates the catalytic activities of DNA polymerase ε (Pol ε). Although pol2-16 mutants survive, they present very tiny spore colonies, increased doubling time, larger than normal cells, aberrant nuclei, and rapid acquisition of suppressor mutations. These phenotypes reveal a severe growth defect that is distinct from that of strains that lack only Pol ε proofreading (pol2-4), consistent with the idea that Pol ε is the major leading-strand polymerase used for unstressed DNA replication. Ribonucleotides are incorporated into the pol2-16 genome in patterns consistent with leading-strand replication by Pol δ when Pol ε is absent. More importantly, ribonucleotide distributions at replication origins suggest that in strains encoding all three replicases, Pol δ contributes to initiation of leading-strand replication. We describe two possible models.
Highlights
To investigate nuclear DNA replication enzymology in vivo, we have studied Saccharomyces cerevisiae strains containing a pol[2,3,4,5,6,7,8,9,10,11,12,13,14,15,16] mutation that inactivates the catalytic activities of DNA polymerase ε (Pol ε)
In order to better understand the consequences of loss of Pol ε catalytic activities on replication in yeast cells, we constructed the pol[2,3,4,5,6,7,8,9,10,11,12,13,14,15,16] mutation and examined its properties
To distinguish between the two replication models, we compared pol[2,3,4,5,6,7,8,9,10,11,12,13,14,15,16] phenotypes in both backgrounds to those of analogous wild-type yeast and to strains lacking Pol ε’s 3′-exonuclease activity due to substitution of alanine for two negatively charged residues (D290 and E292) that are essential for proofreading of mismatches and ribonucleotides[26,27]
Summary
To investigate nuclear DNA replication enzymology in vivo, we have studied Saccharomyces cerevisiae strains containing a pol[2,3,4,5,6,7,8,9,10,11,12,13,14,15,16] mutation that inactivates the catalytic activities of DNA polymerase ε (Pol ε). S. cerevisiae pol21621–23 and S. pombe cdc20ΔN-term[24] mutant strains are viable despite having in-frame deletions of Pol ε polymerase and exonuclease domains while leaving intact the C-terminal domain that controls cellular responses to DNA damage These facts are consistent with an alternative model that is supported by a recent study[25] proposing that Pol δ is the major replicase for both strands and that Pol ε proofreads errors made by Pol δ as it replicates the leading DNA strand. We present ribonucleotide incorporation data with variants of Pols α, δ, and ε that strongly support the hypothesis that Pol δ contributes to initiation of leading-strand replication in yeast by synthesizing DNA of both strands at replication origins
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
