Biol. . Finally, our data also define a novel role for the poorly characterized p12 subunit of hPOL . INTRODUCTION The faithful completion of chromosomal DNA replication Tetrahydropapaverine HCl is of crucial importance Tetrahydropapaverine HCl in determining the fidelity with which genetic information is passed from mother to daughter cells. Incomplete replication or the erroneous copying of a damaged DNA template can give rise to genome instability, accumulation of mutations and, in multicellular organisms, to neoplastic transformation (1). Chromosomal DNA replication in eukaryotic cells requires three distinct DNA polymerases named DNA polymerase (POL ), (POL ) and (POL ). POL and POL are required for replication of the leading strand and for completion of lagging strand DNA synthesis. Their respective roles in the replication of leading and lagging strands are still uncertain, though it has been proposed that POL and POL function specifically at the lagging and leading strands of the replication fork, respectively. POL is also involved in different DNA repair pathways as a gap filling enzyme (2). The mammalian POL has been studied extensively as a core enzyme consisting of four subunits named p125, p66, p50 and p12 (3). Two of the subunits form a tightly-associated catalytic heterodimer consisting of the catalytic p125 subunit, which has both 5 to 3 DNA polymerase and 3 to 5 5 exonuclease activities, and p50. The role of the p66 subunit is to bind PCNA, the homotrimeric sliding clamp that functions as a processivity factor for POL during DNA replication (4). A specific role for the p12 subunit has not been identified thus Tetrahydropapaverine HCl far, although it has been shown to interact with the p125 and p50 subunits of POL and Proliferating Cell Antigen (PCNA) (5), and data from DNA replication assays indicate that addition of p12 enhances the DNA polymerizing activity of the enzyme (6). The levels of p12 are regulated by the proteasome through the mechanism that is not dependent upon p12 ubiquitination (7). Apart from PCNA, no HEY2 other interacting protein has been characterized that specifically associates with p12. The RecQ family of DNA helicases represents a group of evolutionarily Tetrahydropapaverine HCl conserved enzymes that are involved in the maintenance of genome stability (8,9). There are five members of this family known in humans called RECQL1, BLM, WRN, RECQL4 and RECQL5. Defects in three of these give rise to defined clinical disorders associated with cancer predisposition and various aspects of premature aging: mutations in the and genes result in Werner’s syndrome (WS) and RothmundCThomson syndrome (RTS), respectively, both of which feature genome instability, predisposition to some types of cancer and the early onset of several aging features. Mutations in the gene cause Bloom’s syndrome (BS), which is also associated with excessive chromosomal instability and a high incidence of cancers of all types. In contrast to WS and RTS, no obvious premature aging has been observed in BS patients (10). Cells derived from BS patients show a 10-fold higher frequency of reciprocal exchanges between sister chromatids (SCEs), as well as excessive chromosome breakage (11). The BLM protein is a DNA structure-specific helicase that unwinds DNA in 3 to 5 5 direction (12), and shows an apparent preference for unwinding of synthetic Tetrahydropapaverine HCl Holliday junctions, G-quadruplex (G4) DNA and D-loop DNA substrates (13,14). These substrates represent different DNA structures that can be formed during DNA replication and homologous recombination (HR) processes. Cell biological studies have shown that BLM is localized in the nucleus of human cells within discrete foci termed promyelocytic leukemia (PML) nuclear bodies (15). BLM also localizes to nucleoli in S-phase cells (16), and to telomeres in cells lacking telomerase (17). On the basis of the aforementioned reports, it has been proposed that BLM functions at the interface of DNA replication and recombination, and facilitates the repair of damaged DNA replication forks (9,18). A large body of evidence implicates BLM in DNA replication. First, DNA replication defects, such as a retarded rate of nascent DNA chain elongation (19) and accumulation of abnormal replication intermediates (20), have been described in BS cells. Second, BLM interacts physically and functionally with several proteins that play important roles during DNA replication, such as replication protein A (RPA) (21), FEN-1 (22) and chromatin assembly.