An extremely important detail of this mechanism surrounds the control of replication initiation at the origin of replication, and therefore, the origin of replication must be defined by working from simple, well-defined eukaryotic systems towards mammalian systems.  The origin of replication will be defined by both the sequences that constitute the origin of replication initiation (cis-acting factors) as well as the proteins that recognize and bind (trans-acting factors) to these sequences.  This lab is approaching the problem of defining the origin of replication from both directions, with one study attempting to determine and map the sequences at a mammalian origin as well as another study attempting to reconstitute the human origin recognition complex, a key part of the proteins the act at the origin of replication initiation.

Having extended our studies to cellular mammalian DNA replication, we seek to define the cis-acting elements that are required for the function of an origin of DNA replication in mammalian chromosomes in vivo. These elements will be identified using a competitive PCR approach to assess initiation activity of putative origins, as well as mutant origins, that have been stably integrated into ectopic sites in chromosomal DNA. We wish to search for DNA binding sites of initiation proteins thought to assemble on origins, as well as other proteins that interact with them, and determine how they activate replication.

For some time, replication of papovaviral DNA in infected cells and in cell-free reactions has proven to be an extremely useful model system and has facilitated the identification and characterization of many of the cellular proteins required for replication through a basic understanding of the mechanism of SV40 DNA replication. One aspect of our research program focuses on the role of protein phosphorylation in regulating the activity of proteins required for the initiation of SV40 viral DNA replication in a cell-free system. The phosphorylation state of the viral initiator protein T antigen, a DNA helicase, controls its ability to unwind DNA from the viral origin in the circular viral DNA.  Further work studies the interaction of purified SV40 T antigen with single-strand DNA binding protein RPA. 

The accuracy with which genetic information is passed from a parent cell to daughter cells depends on a number of proteins involved in DNA replication, repair and recombination. A prerequisite for all these cellular processes is a transient unwinding of the DNA double helix by a class of enzymes called DNA helicases. DNA helicase B (DHB) has been identified in two mammalian species, mouse and human, but not in lower eukaryotes. The exact role of this helicase remains unknown. However, studies of the human enzyme (HDHB) and its mouse homologue suggest that it plays an important role in DNA metabolism. HDHB physically and functionally interacts with DNA replication/repair proteins and dominant-negative mutants of the helicase block the cell cycle at the onset of DNA replication. We employ a combination of biochemical approaches, analysis of dominant-negative mutants in living cells, cell-cycle and tissue-specific regulation of HDHB expression to elucidate role of DNA helicase B in DNA metabolism.

Furthermore, phosphorylation of human DNA polymerase- α primase regulates its replication activity in a cell cycle-dependent manner, and this mechanism also has been under investigation.  Current work suggests that HDHB is involved in DNA damage signal processing and repair.

Another aspect of our research program focusses on the role of protein phosphorylation in regulating the initiation of DNA replication in a cell-free system. We have shown that phosphorylation of human DNA polymerase alpha-primase regulates its ability to synthesize RNA primers in a cell cycle-dependent manner. We are now testing the hypothesis that phosphate turnover on one of the polymerase-primase subunits responds to DNA damage/checkpoint signals.

DNA replication, repair, and recombination all require a DNA helicase activity to separate the two DNA strands and permit DNA processing. We have cloned a cDNA encoding a novel human DNA helicase that is required for the onset of chromosomal DNA replication. The function of the helicase is not known and orthologs have not been identified in lower eukaryotic genomes so far. The helicase appears in a focal pattern in the nucleus in G1, and most of it is exported to the cytoplasm in S phase. We have shown that the helicase interacts physically and functionally with DNA polymerase alpha-primase and replication protein A. Current work seeks to identify additional proteins that interact with the helicase and to elucidate its function using genetic and biochemical methods.

Mammalian chromosomes contain about 40,000 origins that direct initiation of chromosomal DNA at specific times in S phase. However, the cis-acting elements required to constitute a functional origin of DNA replication in mammalian chromosomes in vivo are not known. To address this question, we use a competitive PCR approach to assess initiation activity of a mammalian origin that has been stably integrated into ectopic sites in chromosomal DNA, as well as mutant versions of the origin. The ectopic origin is active in multiple chromosomal sites. Three essential sequence-specific elements in the origin have been identified so far. We have mapped the binding sites of initiation proteins in the functional origin and in mutant origins by chromatin immunoprecipitation. Future work will determine how initiation protein binding and chromatin structure cooperate to activate replication.