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2004 Scientific Report

Laboratory of Chromosome

Laboratory of Chromosome Replication Michael Weinreich, Ph.D. Dr. Weinreich received his Ph.D. in biochemistry from the University of Wisconsin–Madison in 1993. He then served as a postdoctoral fellow in the laboratory of Bruce Stillman, director of the Cold Spring Harbor Laboratory, New York, from 1993 to 2000. Dr. Weinreich joined VARI as a Scientific Investigator in March 2000. Staff Don Pappas, Ph.D. Hongyu Liu, M.S. Aaron DeWard, B.S. Ryan Frisch, B.S. Carrie Gabrielse, B.S. Laboratory Members Students Aaron DeWard Victoria Hammond Kelli VanDussen Research Interests We are interested in how the initiation of chromosomal DNA replication occurs in budding yeast and in human cells. When cells reach a critical size in G1 phase and receive the appropriate growth signals, they commit to the process of cell division. The first essential step following commitment is the duplication of the genome, which occurs during S phase. Therefore, the transition from G1 into S phase (or from quiescence into the cell division cycle) is a highly regulated event. The initiation of DNA synthesis occurs at multiple replication origins throughout the genome during S phase, but each replication origin is activated only once per cell cycle. The timing and frequency of initiation is precisely controlled because errors in this process could cause genome amplification and instability. Since uncontrolled cell division and genomic instability are properties of many cancer cells, we are also investigating the aberrant regulation of initiation factors in the development of cancer. The initiation of DNA synthesis requires at least four steps (Fig. 1). The first is origin marking by ORC, a six-subunit protein complex that is required for replication initiation and that recognizes conserved DNA sequence elements in all origins. ORC then directs the assembly of a large macromolecular complex called the “pre-replicative complex,” or pre-RC. As cells exit mitosis, the Cdc6 protein binds to ORC and, together with Cdt1p, promotes loading of the MCM helicase at origins. ORC, Cdc6p, Cdt1p, and the MCM complex are together required to form the pre-RC. Additional proteins associate with the pre-RC during G1 to form a “pre-initiation complex”, and then this large complex of proteins is activated to form bi-directional replication forks by the two-subunit Cdc7p-Dbf4p protein kinase. The processes of origin unwinding and the recruitment of DNA polymerases are not well understood. We are focusing on how Cdc6p promotes pre- RC assembly and how the Cdc7p-Dbf4p kinase triggers initiation. Cdc6p is a critical, limiting factor for assembly of the pre-RC. We have previously shown that Cdc6p interacts with ORC and that its essential activity requires a functional ATP-binding domain. We have taken a genetic approach to identify genes that act together with CDC6 and have defined several previously unknown genes that influence DNA replication. Figure 1. The yeast chromosome replication cycle 56

WT cdc6-4 sir2-N345A cdc6-4 sir2-N345A 25ºC 37ºC Figure 2. A catalytic mutation in SIR2 suppresses the cdc6- 4 temperature sensitivity. Serial tenfold dilutions of wild type (WT), cdc6-4, sir2-N345A, and cdc6-4 sir2-N345A cultures were spotted onto rich medium at 25 ˚C and 37 ˚C to assay for growth. The cdc6-4 strain is unable to grow at 37 ˚C but grows as WT following inactivation of Sir2 enzymatic function using the N345A mutation. For instance, we identified loss-of-function mutations in SIR2—which encodes a histone deacetylase required for the formation of transcriptionally “silent” heterochromatin—that suppress a cdc6 temperature-sensitive mutant (Fig. 2). We have further demonstrated that Sir2p is acting in a novel pathway to negatively regulate initiation at some (but not all) origins in yeast. The loss of Sir2p promotes MCM loading at origins when Cdc6p activity is compromised (Fig. 3), suggesting that Sir2p may directly influence chromatin structure at some origins or may regulate the expression or activity of a critical initiation protein. Another of our efforts is to understand how the Cdc7p-Dbf4p serine/threonine kinase triggers replication initiation in yeast and in human cells. Cdc7p kinase requires the Dbf4p subunit for activity, which is periodically degraded during the cell cycle. We have purified the human and yeast Cdc7p-Dbf4p kinases from baculovirus-infected Sf9 cells and have previously shown that the yeast protein phosphorylates subunits of the MCM complex and also DNA polymerase α-primase, the initiating polymerase. We are examining the substrate specificity of the Cdc7p-Dbf4p kinase in an effort to determine its essential targets for DNA replication initiation. Dbf4p itself is phosphorylated in a MEC1- and RAD53-dependent manner following inhibition of DNA replication, and this appears to inhibit Cdc7p-Dbf4p kinase activity. Mec1p is a homologue of the human ATM/ATR kinases that are key regulators of the response to DNA damage. Rad53p is the homologue of the human CHK2 checkpoint kinase. Genetic data also suggest that Cdc7p-Dbf4p kinase is required for recovery from replication-induced arrest or DNA damage. We have shown by a structure-function analysis that an amino-terminal BRCT domain within Dbf4p is required for the proper response to a variety of DNA-damaging agents. The BRCT domain is a phosphopeptide binding module that was first discovered in the BRCA1 breast cancer susceptibility gene, and it is present in a variety of proteins involved in the response to DNA damage. The Dbf4p BRCT domain is, however, not A B PCR products 350bp 310bp 270bp ARS1 382bp 332bp 302bp 272bp ARS315 228bp 15kb 15kb 231bp 272bp 309bp 346bp 25kb ARS501 G2/M arrest 25 o C (Nocodazole) MCM loading Release a-factor Shift to 37 o C, 30' (Noc) 90', 120' MCM loading Release a-factor 37 o C 60', 90' C D Wild Type cdc6-4 cdc6-4 sir2∆ Input DNA noc 90' 120' noc 90' 120' noc 90' 120' Mcm2p ChIP - 25 o C Wild Type cdc6-4 cdc6-4 sir2∆ Input DNA noc 60' 90' noc 60' 90' noc 60' 90' Mcm2p ChIP - 37 o C ARS1 ARS315 ARS501 ARS1 ARS315 ARS501 Figure 3. Deletion of SIR2 restores Mcm2p loading at origins in the cdc6-4 mutant. Mcm2p chromatin immunoprecipitation (ChIP) assays were performed using cells arrested in G2/M with nocodazole and then released to a G1 arrest for 60, 90, or 120 min, to allow MCM protein loading at origins. The location of PCR primers shown in (A) were used to amplify origin and nearby nonorigin sequences for the ARS1, ARS315, and ARS501 origins following immunoprecipitation of Mcm2p. Data for the cdc6-4 permissive (C) and nonpermissive temperature (D) is shown. A representative input DNA PCR sample is shown for each origin examined. 57

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