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2007 Scientific Report

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VARI |

VARI | 2007 Research Interests We are studying how cells accurately replicate their DNA, a process that begins at specific DNA sequences termed replication origins. There are approximately 400 replication origins in budding yeast and as many as 10,000 in human cells. Coordinating the activation of these origins for DNA synthesis during the cell cycle is a daunting task. We know that origins recruit many proteins prior to the DNA synthetic period (S-phase) that are required for the assembly and activation of replication forks. These proteins include Cdt1p, Cdc6p, and the origin recognition complex (ORC), which binds directly to origin DNA. Cdt1p, Cdc6p, and ORC cooperate to load the MCM DNA helicase at the origin in an ATP-dependent reaction. There are perhaps a score of additional proteins that assemble at the origin following MCM loading before DNA synthesis can begin. In our lab we are studying how Cdc6p activity is influenced by chromatin structure and ATP binding. We previously isolated genetic suppressors of a cdc6-4 temperature-sensitive (ts) mutant that inactivated the SIR2 gene. Sir2p is a histone H3 and H4 deacetylase, and therefore its loss leads to increased H3 and H4 acetylation within chromatin. Although loss of SIR2 allowed growth of the cdc6-4 strain at high temperature, we have found that Sir2p inhibits only specific origins. We have systematically identified multiple SIR2-regulated origins on chromosomes III and VI. Our studies so far indicate that these origins share a common organization including an inhibitory element through which Sir2p acts. Origins in Saccharomyces cerevisiae have a modular structure (Fig. 1) that includes an ORC binding site (A and B1 elements) and a loading site for the MCM helicase (B2 element). We have identified an inhibitory sequence in SIR2-regulated origins, termed the I S element, located downstream of B2. This element is responsible for Sir2p inhibition at these origins. Recent high-resolution mapping along chromosome III indicates that the I S element maps squarely within a positioned nucleosome. This nucleosome is directly adjacent to or overlaps the B2 element and therefore might influence MCM helicase loading. In support of this, we have found that excluding this nucleosome from the B2 element abolishes the activity of the I S element. Furthermore, the I S element acts in a distance-dependent manner, which is consistent with an effect through this positioned nucleosome. 75 Figure 1. Figure 1. S. cerevisiae origins have a modular structure consisting of an essential A element and important B elements. These elements direct binding of proteins in the “pre-replicative complex” (pre-RC) that forms during G1 phase. An inhibitory element (I S ) is present at some origins and likely interferes with pre-RC assembly.

Van Andel Research Institute | Scientific Report How might SIR2 inhibit DNA replication? We believe that this occurs through deacetylation of histone H4 K16. Sir2p deacetylates the histone H3 acetyl-lysine residues K9 and K14 as well as H4 K16. We found that an H4 mutation of K16 to Q that mimics the acetylated state suppresses the cdc6-4 and mcm2-1 ts mutations; H3 K9Q or K14Q mutations do not suppress these ts mutations. Taken together, our data suggest that a local nucleosome acetylated on H4 K16 facilitates MCM helicase loading and that a nucleosome impinging on B2, if it is deacetylated on K16, inhibits MCM loading. We would like to understand the molecular function of the I S element, which is presumably affecting this histone H4 modification. Based on the frequency of SIR2-regulated origins on chromosome III and VI, we expect that a significant number of origins (about 80 of 400) will be subject to this type of regulation. 76 The Cdc7p-Dbf4p kinase promotes DNA replication and assists in repair of certain DNA lesions. Cdc7p-Dbf4p is a two-subunit serine/threonine kinase required for initiating DNA replication, and it acts after assembly of the MCM helicase as diagramed in Fig 2. Cdc7p is the kinase subunit but it has no activity in the absence of the Dbf4p regulatory subunit. We have analyzed Dbf4p using a structure-function approach to determine the residues required for its essential role in DNA replication. We found that about 40% of the Dbf4p N-terminus is dispensable for its essential replication function, but that it encodes a conserved 100- amino-acid region with similarity to the BRCT motif. We have called this sequence the BRDF motif for BRCT and DBF4 similarity. The BRCT domain folds into a modular structure and is often found in proteins that participate in the DNA damage response. The BRCT domain likely binds to phosphoproteins and therefore allows regulated targeting to proteins modified by phosphorylation, as occurs following activation of the DNA damage checkpoint. Yeast dbf4 mutants altering this motif are sensitive to replication fork arrest, suggesting that the BRDF domain targets the kinase to stalled replication forks (Fig. 3). In support of this interpretation, we have performed domain-swapping experiments and identified a heterologous BRCT domain that will function in place of the Dbf4p BRDF domain. We are testing whether these two domains target Dbf4p to the same or similar substrates. It appears therefore, that the Dbf4p BRDF motif has a role in maintaining replication fork stability, likely through targeting Cdc7p kinase to non-origin sites. This is a separable activity to the essential role of Dbf4p in promoting the initiation of replication. Figure 2. Figure 2. DNA synthesis requires Cdc7p-Dbf4p kinase, which is thought to act on the pre-RC to promote Cdc45p and GINS binding. Assembly of a “pre-initiation complex” facilitates origin unwinding to give ssDNA. Figure 3. A. Figure 3. A) Schematic representation showing elements conserved among all Dbf4p orthologs. The N-terminal BRDF domain is dispensable for DNA replication. B) We propose this BRCT-like domain directs Cdc7p-Dbf4p kinase to stalled replication forks via recognition of a phosphorylated protein in the replisome. B.

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