Recruitment of DNA repair synthesis machinery to sites of DNA damage/repair in living human cells

The eukaryotic sliding DNA clamp, proliferating cell nuclear antigen (PCNA), is essential for DNA replication and repair synthesis. In order to load the ring-shaped, homotrimeric PCNA onto the DNA double helix, the ATPase activity of the replication factor C (RFC) clamp loader complex is required. Although the recruitment of PCNA by RFC to DNA replication sites has well been documented, our understanding of its recruitment during DNA repair synthesis is limited. In this study, we analyzed the accumulation of endogenous and fluorescent-tagged proteins for DNA repair synthesis at the sites of DNA damage produced locally by UVA-laser micro-irradiation in HeLa cells. Accumulation kinetics and in vitro pull-down assays of the large subunit of RFC (RFC140) revealed that there are two distinct modes of recruitment of RFC to DNA damage, a simultaneous accumulation of RFC140 and PCNA caused by interaction between PCNA and the extreme N-terminus of RFC140 and a much faster accumulation of RFC140 than PCNA at the damaged site. Furthermore, RFC140 knock-down experiments showed that PCNA can accumulate at DNA damage independently of RFC. These results suggest that immediate accumulation of RFC and PCNA at DNA damage is only partly interdependent.     

A conserved interaction between the replicative clamp loader and DNA ligase in eukaryotes: implications for Okazaki fragment joining

The recruitment of DNA ligase I to replication foci and the efficient joining of Okazaki fragments is dependent on the interaction between DNA ligase I and proliferating cell nuclear antigen (PCNA). Although the PCNA sliding clamp tethers DNA ligase I to nicked duplex DNA circles, the interaction does not enhance DNA joining. This suggests that other factors may be involved in the joining of Okazaki fragments. In this study, we describe an association between replication factor C (RFC), the clamp loader, and DNA ligase I in human cell extracts. Subsequently, we demonstrate that there is a direct physical interaction between these proteins that involves both the N- and C-terminal domains of DNA ligase I, the N terminus of the large RFC subunit p140, and the p36 and p38 subunits of RFC. Although RFC inhibited DNA joining by DNA ligase I, the addition of PCNA alleviated inhibition by RFC. Notably, the effect of PCNA on ligation was dependent on the PCNA-binding site of DNA ligase I. Together, these results provide a molecular explanation for the key in vivo role of the DNA ligase I/PCNA interaction and suggest that the joining of Okazaki fragments is coordinated by pairwise interactions among RFC, PCNA, and DNA ligase I.     

The large subunit of replication factor C is a substrate for caspase-3 in vitro and is cleaved by a caspase-3-like protease during Fas-mediated apoptosis.

Caspase-3 is an ICE-like protease activated during apoptosis induced by different stimuli. Poly(ADP-ribose) polymerase (PARP), the first characterized substrate of caspase-3, shares a region of homology with the large subunit of Replication Factor C (RF-C), a five-subunit complex that is part of the processive eukaryotic DNA polymerase holoenzymes. Caspase-3 cleaves PARP at a DEVD-G motif present in the 140 kDa subunit of RF-C (RFC140) and evolutionarily conserved. We show that cleavage of RFC140 during Fas-mediated apoptosis in Jurkat cells and lymphocytes results in generation of multiple fragments. Cleavage is inhibited by the caspase-3-like protease inhibitor Ac-DEVD-CHO but not the caspase-1/ICE-type protease inhibitor Ac-YVAD-CHO. In addition, recombinant caspase-3 cleaves RFC140 in vitro at least at three different sites in the C-terminal half of the protein. Using amino-terminal microsequencing of radioactive fragments, we identified three sites: DEVD723G, DLVD922S and IETD1117A. We did not detect cleavage of small subunits of RF-C of 36, 37, 38 and 40 kDa by recombinant caspase-3 or by apoptotic Jurkat cell lysates. Cleavage of RFC140 during apoptosis inactivates its function in DNA replication and generates truncated forms that further inhibit DNA replication. These results identify RFC140 as a critical target for caspase-3-like proteases and suggest that caspases could mediate cell cycle arrest.     

Regulation of RelA Subcellular Localization by a Putative Nuclear Export Signal and p50

Nuclear factor kappaB (NF-kappaB) represents a family of dimeric DNA binding proteins, the pleotropic form of which is a heterodimer composed of RelA and p50 subunits. The biological activity of NF-kappaB is controlled through its subcellular localization. Inactive NF-kappaB is sequestered in the cytoplasm by physical interaction with an inhibitor, IkappaBalpha. Signal-mediated IkappaBalpha degradation triggers the release and subsequent nuclear translocation of NF-kappaB. It remains unknown whether the NF-kappaB shuttling between the cytoplasm and nucleus is subjected to additional steps of regulation. In this study, we demonstrated that the RelA subunit of NF-kappaB exhibits strong cytoplasmic localization activity even in the absence of IkappaBalpha inhibition. The cytoplasmic distribution of RelA is largely mediated by a leucine-rich sequence homologous to the recently characterized nuclear export signal (NES). This putative NES is both required and sufficient to mediate cytoplasmic localization of RelA as well as that of heterologous proteins. Furthermore, the cytoplasmic distribution of RelA is sensitive to a nuclear export inhibitor, leptomycin B, suggesting that RelA undergoes continuous nuclear export. Interestingly, expression of p50 prevents the cytoplasmic expression of RelA, leading to the nuclear accumulation of both RelA and p50. Together, these results suggest that the nuclear and cytoplasmic shuttling of RelA is regulated by both an intrinsic NES-like sequence and the p50 subunit of NF-kappaB.