Biomedical subjects

Alan E Tomkinson

Publications and source records attributed to Alan E Tomkinson.

21 records · Page 2Linked to original sources

PubMed2003

The human checkpoint Rad protein Rad17 is chromatin-associated throughout the cell cycle, localizes to DNA replication sites, and interacts with DNA polymerase epsilon.

The checkpoint Rad proteins Rad17, Rad9, Rad1, Hus1, ATR, and ATRIP become associated with chromatin in response to DNA damage caused by genotoxic agents and replication inhibitors, as well as during unperturbed DNA replication in S phase. Here we show that murine Rad17 is phosphorylated at two sites that were previously shown to be modified in response to DNA damage, independent of DNA damage and ATM, in proliferating tissue. In contrast to studies with Xenopus laevis extracts but similar to observations in Schizosaccharomyces pombe, the level of chromatin-bound hRad17 remains relatively constant during the cell cycle and does not change significantly in response to DNA damage or replication block. However, phosphorylated hRad17 preferentially associates with the sites of ongoing DNA replication and interacts with the DNA replication protein, DNA polymerase epsilon. These results provide a link between the DNA damage checkpoint machinery and the replication apparatus and suggest that hRad17 may play a role in monitoring the progress of DNA replication via its interaction with DNA polymerase epsilon.

Animals↗

PubMed2003

Physical and functional interaction between DNA ligase IIIalpha and poly(ADP-Ribose) polymerase 1 in DNA single-strand break repair.

The repair of DNA single-strand breaks in mammalian cells is mediated by poly(ADP-ribose) polymerase 1 (PARP-1), DNA ligase IIIalpha, and XRCC1. Since these proteins are not found in lower eukaryotes, this DNA repair pathway plays a unique role in maintaining genome stability in more complex organisms. XRCC1 not only forms a stable complex with DNA ligase IIIalpha but also interacts with several other DNA repair factors. Here we have used affinity chromatography to identify proteins that associate with DNA ligase III. PARP-1 binds directly to an N-terminal region of DNA ligase III immediately adjacent to its zinc finger. In further studies, we have shown that DNA ligase III also binds directly to poly(ADP-ribose) and preferentially associates with poly(ADP-ribosyl)ated PARP-1 in vitro and in vivo. Our biochemical studies have revealed that the zinc finger of DNA ligase III increases DNA joining in the presence of either poly(ADP-ribosyl)ated PARP-1 or poly(ADP-ribose). This provides a mechanism for the recruitment of the DNA ligase IIIalpha-XRCC1 complex to in vivo DNA single-strand breaks and suggests that the zinc finger of DNA ligase III enables this complex and associated repair factors to locate the strand break in the presence of the negatively charged poly(ADP-ribose) polymer.

Cell Nucleus↗

PubMed2002

A physical and functional interaction between yeast Pol4 and Dnl4-Lif1 links DNA synthesis and ligation in nonhomologous end joining.

Genetic studies have implicated the Saccharomyces cerevisiae POL4 gene product in the repair of DNA double-strand breaks by nonhomologous end joining. Here we show that Pol4 preferentially catalyzes DNA synthesis on small gaps formed by the alignment of linear duplex DNA molecules with complementary ends, a DNA substrate specificity that is compatible with its predicted role in the repair of DNA double-strand breaks. Pol4 also interacts directly with the Dnl4 subunit of the Dnl4-Lif1 complex via its N-terminal BRCT domain. This interaction stimulates the DNA synthesis activity of Pol4 and, to a lesser extent, the DNA joining activity of Dnl4-Lif1. Notably, the joining of DNA substrates that require the combined action of Pol4 and Dnl4-Lif1 is much more efficient than the joining of similar DNA substrates that require only ligation. Thus, the physical and functional interactions between Pol4 and Dnl4-Lif1 provide a molecular mechanism for both the recruitment of Pol4 to in vivo DNA double-strand breaks and the coupling of the gap filling DNA synthesis and DNA joining reactions that complete the microhomology-mediated pathway of nonhomologous end joining.

DNA↗