Joseph L. Stodola

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The eukaryotic replicative DNA polymerases (Pol α, δ and ɛ) and the major DNA mutagenesis enzyme Pol ζ contain two conserved cysteine-rich metal-binding motifs (CysA and CysB) in the C-terminal domain (CTD) of their catalytic subunits. Here we demonstrate by in vivo and in vitro approaches the presence of an essential [4Fe-4S] cluster in the CysB motif of(More)
DNA polymerase ζ (Pol ζ) plays a key role in DNA translesion synthesis (TLS) and mutagenesis in eukaryotes. Previously, a two-subunit Rev3-Rev7 complex had been identified as the minimal assembly required for catalytic activity in vitro. Herein, we show that Saccharomyces cerevisiae Pol ζ binds to the Pol31 and Pol32 subunits of Pol δ, forming a(More)
VACM-1, a cul5 gene product, when overexpressed in vitro, has an antiproliferative effect. In vivo, VACM-1/cul5 is present in tissues involved in the regulation of water balance. Neither proteins targeted for VACM-1/cul5-specific degradation nor factors that may regulate its expression in those tissues have been studied. To identify genes that may be(More)
The homotrimeric sliding clamp proliferating cell nuclear antigen (PCNA) mediates Okazaki fragment maturation through tight coordination of the activities of DNA polymerase δ (Pol δ), flap endonuclease 1 (FEN1) and DNA ligase I (Lig1). Little is known regarding the mechanism of partner switching on PCNA and the involvement of PCNA's three binding sites in(More)
DNA polymerase delta (Pol δ) is responsible for elongation and maturation of Okazaki fragments. Pol δ and the flap endonuclease FEN1, coordinated by the PCNA clamp, remove RNA primers and produce ligatable nicks. We studied this process in the Saccharomyces cerevisiae machinery at millisecond resolution. During elongation, PCNA increased the Pol δ catalytic(More)
The strand displacement activity of DNA polymerase δ is strongly stimulated by its interaction with proliferating cell nuclear antigen (PCNA). However, inactivation of the 3'-5' exonuclease activity is sufficient to allow the polymerase to carry out strand displacement even in the absence of PCNA. We have examined in vitro the basic biochemical properties(More)
Using a DNA polymerase coupled assay and FRET (Förster resonance energy transfer)-based helicase assays, in this work, we show that a monomer of Saccharomyces cerevisiae Pif1 can unwind dsDNA (double-stranded DNA). The helicase activity of a Pif1 monomer is modulated by the nature of the 3'-ssDNA (single-stranded DNA) tail of the substrate and its effect on(More)
DNA replication in eukaryotic cells requires minimally three B-family DNA polymerases: Pol α, Pol δ, and Pol ϵ. Pol δ replicates and matures Okazaki fragments on the lagging strand of the replication fork. Saccharomyces cerevisiae Pol δ is a three-subunit enzyme (Pol3-Pol31-Pol32). A small C-terminal domain of the catalytic subunit Pol3 carries both(More)
Primer29 5’-TCA GCG CGA GCA TGA CAT TGA AGG TAA CC-3’ Primer29-Cy3 5’-Cy3-TCA GCG CGA GCA TGA CAT TGA AGG TAA CC-3’ Template-AT20 5’-BiotinTEG-TTC CTT CAA CCA GCT TAC CTT CTT CCT TTT TTT TTT TTT TTT TTT TAG GTT ACC TTC AAT GTC ATG CTC GCG CTG A-BiotinTEG-3’ Template-Sub I 5’-BiotinTEG-TCT TCC TTC AAC CAG CTT ACC TTC TTC CTT TTA GGT TAC CTT CAA TGT CAT GCT(More)
Using an in vitro reconstituted system in this work we provide direct evidence that the yeast repressor/activator protein 1 (Rap1), tightly bound to its consensus site, forms a strong non-polar barrier for the strand displacement activity of DNA polymerase . We propose that relief of inhibition may be mediated by the activity of an accessory helicase. To(More)