Topoisomerase II␤-binding protein (TopBP1), a human protein with eight BRCT domains, is similar to Saccharomyces cerevisiae Dpb11 and Schizosaccharomyces pombe Cut5 checkpoint proteins and closely related to Drosophila Mus101. We show that human TopBP1 is required for DNA replication and that it interacts with DNA polymerase ⑀. In S phase TopBP1 colocalizes with Brca1 to foci that do not represent sites of ongoing DNA replication. Inhibition of DNA synthesis leads to relocalization of TopBP1 together with Brca1 to replication forks, suggesting a role in rescue of stalled forks. DNA damage induces formation of distinct TopBP1 foci that colocalize with Brca1 in S phase, but not in G 1 phase. We also show that TopBP1 interacts with the checkpoint protein hRad9. Thus, these results implicate TopBP1 in replication and checkpoint functions. DNA polymerases (pol)1 play essential roles in chromosomal DNA replication and repair. In Saccharomyces cerevisiae three essential nuclear polymerases, ␣, ␦, and ⑀ have important functions in DNA replication. S. cerevisiae pol ⑀ is isolated as a complex of a catalytic subunit and three smaller subunits, Dpb2, 3, and 4 (1). This four-subunit structure is also conserved in the human enzyme, which consists of a catalytic subunit (2), a B subunit (3, 4), and two smaller subunits (5). Pol ⑀ is a proofreading DNA polymerase, which has been implicated in DNA replication, as temperature-sensitive mutants show defects in DNA replication in both S. cerevisiae and Schizosaccharomyces pombe (6 -8). Moreover, pol ⑀ is associated with origins of DNA replication and it proceeds along the replication fork (9). In human cells, pol ⑀ is associated with actively replicated cellular DNA (10) and has been shown to perform an important fraction of replicative DNA synthesis (11). Surprisingly, the catalytic domain of pol ⑀ is not essential for viability in S. cerevisiae. Instead, the C terminus, which interacts with Dpb2, exerts all of the essential functions (12).Pol ⑀ has been proposed to function in the repair of UVdamaged DNA because it is able to catalyze UV-induced DNA synthesis in vivo (13) and performs efficient gap-filling synthesis in the reconstituted nucleotide excision repair system (14). A role in base excision repair is suggested by the fact that pol ⑀ mutants fail to support repair synthesis in vitro, and repair activity can be restored by the addition of purified pol ⑀ (15). Pol ⑀ has also been proposed to function in a specialized replication process required to repair double strand breaks (16). In addition to replicative and repair roles, it has been suggested that pol ⑀ coordinates transcriptional and cell cycle responses to DNA damage and replication blocks (17).In S. cerevisiae, a BRCT domain-containing protein, Dpb11, interacts with the pol ⑀ complex and was originally identified as a suppressor of pol ⑀ catalytic and Dpb2 subunit mutants (18,19). DPB11 is an essential gene required for DNA replication (18). The inability of DPB11 mutants to restrain mitosis in the presence of inco...

DNA polymerases (pols) have a central role in DNA replication and maintenance of chromosomal DNA [1]. At least 14 pols have been identified in the mammalian cell, but only three -pols a, d and e -are needed to synthesize the bulk of DNA during nuclear DNA replication. These pols are structurally related, belonging to the family B DNA polymerases [2]. Nonetheless, all three perform additional roles in other DNA transactions as well as transduce signals of cell cycle control and DNA damage response [1].Only pol a is capable of initiating DNA synthesis de novo owing to its associated primase activity [3]. The major function of pol a ⁄ primase is synthesizing a short RNA-DNA primer of 30-40 nucleotides that serves both to initiate leading strand DNA replication and to provide precursors of the 200 nucleotide-long Okazaki fragments on the lagging strand [4][5][6]. Pol a ⁄ primase is then replaced by the elongating pols d or e. This switch from pol a to pol d is controlled by replication factor C, which loads the processivity factor, Keywords cell cycle; DNA polymerase; DNA replication; electron microscopy; UV crosslinking The contributions of human DNA polymerases (pols) a, d and e during S-phase progression were studied in order to elaborate how these enzymes co-ordinate their functions during nuclear DNA replication. Pol d was three to four times more intensely UV cross-linked to nascent DNA in late compared with early S phase, whereas the cross-linking of pols a and e remained nearly constant throughout the S phase. Consistently, the chromatin-bound fraction of pol d, unlike pols a and e, increased in the late S phase. Moreover, pol d neutralizing antibodies inhibited replicative DNA synthesis most efficiently in late S-phase nuclei, whereas antibodies against pol e were most potent in early S phase. Ultrastructural localization of the pols by immuno-electron microscopy revealed pol e to localize predominantly to ring-shaped clusters at electron-dense regions of the nucleus, whereas pol d was mainly dispersed on fibrous structures. Pol a and proliferating cell nuclear antigen displayed partial colocalization with pol d and e, despite the very limited colocalization of the latter two pols. These data are consistent with models where pols d and e pursue their functions at least partly independently during DNA replication.Abbreviations BrdU, bromodeoxyuridine; CLSM, confocal laser-scanning microscopy; EM, electron microscopy; immuno-EM, immuno electron microscopy; MCM2, minichromosome maintenance 2; NP-40, Nonidet P-40; PCNA, proliferating cell nuclear antigen; pol, DNA polymerase.

Background: Replicative DNA polymerases ␦ and ⑀ are believed to synthesize lagging and leading strands, respectively. Results: Human DNA polymerases ␣/␦ and ⑀ segregate during S phase and DNA polymerase ⑀ alone remains bound to lamins. Conclusion: DNA polymerases ␦ and ⑀ act independently in late S phase Significance: Human cell DNA replication may mechanistically differ from prokaryotic replication.

RNA polymerase II (RNA pol II) transcribes proteinencoding genes in eukaryotes. It can be purified as a 'core' enzyme containing 10-12 subunits with a molecular mass of 500 kDa. However, larger RNA pol II-containing complexes, capable of transcribing from model promoters in vitro with minimal addition of general transcription factors, have also been purified [1]. These 'holoenzyme' complexes contain general transcription factors, other transcriptional mediators, as well as various sets of accessory proteins, such as chromatin remodelling factors [2]. The carboxy-terminal domain (CTD) of RNA pol II has been implicated in mediating interactions with other factors involved in transcription and mRNA processing, and appears to be a major target of regulation. The CTD comprises tandem heptapeptide repeats of the DNA polymerase e co-operates with polymerases a and d in the replicative DNA synthesis of eukaryotic cells. We describe here a specific physical interaction between DNA polymerase e and RNA polymerase II, evidenced by reciprocal immunoprecipitation experiments. The interacting RNA polymerase II was the hyperphosphorylated IIO form implicated in transcriptional elongation, as inferred from (a) its reduced electrophoretic mobility that was lost upon phosphatase treatment, (b) correlation of the interaction with phosphorylation of Ser5 of the C-terminal domain heptapeptide repeat, and (c) the ability of C-terminal domain kinase inhibitors to abolish it. Polymerase e was also shown to UV crosslink specifically a-amanitin-sensitive transcripts, unlike DNA polymerase a that crosslinked only to RNA-primed nascent DNA. Immunofluorescence microscopy revealed partial colocalization of RNA polymerase IIO and DNA polymerase e, and immunoelectron microscopy revealed RNA polymerase IIO and DNA polymerase e in defined nuclear clusters at various cell cycle stages. The RNA polymerase IIO-DNA polymerase e complex did not relocalize to specific sites of DNA damage after focal UV damage. Their interaction was also independent of active DNA synthesis or defined cell cycle stage.Abbreviations BrdU, bromodeoxyuridine; CTD, carboxyterminal domain; DRB, 5,6-dichloro-1-beta-D-ribobenzimidazole; Pol, DNA polymerase; RNA pol II, RNA polymerase II; TFIIH, transcription factor II H.

High fidelity of genome duplication is ensured by cooperation of polymerase proofreading and mismatch repair (MMR) activities. Here, we show that human mismatch recognizing proteins MutS homolog 2 (MSH2) and MSH6 copurify and interact with replicative Pol α. This enzyme also is the replicative primase and replicates DNA with poor fidelity. We show that MSH2 associates with known human replication origins with different dynamics than DNA polymerase (Pol α). Furthermore, we explored the potential functional role of Pol α in the mismatch repair reaction using an in vitro mismatch repair assay and observed that Pol α promotes mismatch repair. Taken together, we show that human Pol α interacts with MSH2-MSH6 complex and propose that this interaction occurs during the mismatch repair reaction.