DNA replication machinery of the mammalian cell

The Human Breast Cancer DNA Synthesome Can Serve as a Novel In Vitro Model System for Studying the Mechanism of Action of Anticancer Drugs

“…Clinical trials have shown that dFdC is effective in most solid tumors and more potent and less toxic than araC [11,13,14,19,21]. Studies of intact cells have indicated that inhibition of DNA synthesis is the predominant effect of dFdC and araC [16,17,26,27,29]. Like araC, the major targets for dFdCTP are the DNA polymerases.…”

“…Full-length DNA was produced in the presence of very low levels of both drugs, suggesting that incorporation of dFdCTP and araCTP did not stop the polymerases from elongating the DNA template. Our results were in agreement with those of Ross et al [29], who demonstrated that dFdC was progressively incorporated into nascent DNA of increasing size in intact HL60 cells. In contrast, studies of dFdCTP incorporation using in vitro primer extension assays by purified DNA polymerase a and 8 demonstrated that after incorporation of dFdCTP to the 3' terminus of the elongating DNA strand, one more deoxynucleotide can be added before the DNA polymerases are unable to continue elongating the nascent strand.…”

DNMT1 is a Component of a Multiprotein DNA Replication Complex

“…32 Poly (ADP-ribose) polymerase has also been identified in the complex, and several proteins within the complex are ADP-ribosylated suggesting that this enzyme may function in a regulatory capacity. 38 Recently the Werner syndrome helicase was also identified.…”

“…The Malkas-Hickey laboratories have previously reported on the isolation and extensive characterization of a multiprotein replication complex from human and mouse cells that fully supports origin directed, semi-conservative DNA replication in a cell-free system, termed the DNA 'synthesome'. 32,33 A number of proteins known to be required for eukaryotic DNA synthesis in vitro have been shown to copurify with the synthesome, including DNA polymerases α, δ and ε, DNA primase, replication protein A (RPA), replication factor-C (RF-C), DNA ligase I, topoisomerases I and II, and PCNA. 32,[34][35][36][37] Other proteins that function in a regulatory capacity such as poly(ADP-ribose) polymerase (PARP), have also been identified in the complex.…”

“…32,33 A number of proteins known to be required for eukaryotic DNA synthesis in vitro have been shown to copurify with the synthesome, including DNA polymerases α, δ and ε, DNA primase, replication protein A (RPA), replication factor-C (RF-C), DNA ligase I, topoisomerases I and II, and PCNA. 32,[34][35][36][37] Other proteins that function in a regulatory capacity such as poly(ADP-ribose) polymerase (PARP), have also been identified in the complex. 38 In the present study, we show that DNMT1 protein and activity copurify with the DNA synthesome isolated from human cells.…”