The long-term goal of our research is to elucidate the regulation and mechanism of eukaryotic DNA replication. This essential process requires complex coordination to produce an exact copy of the genome every cell cycle. Central to the regulation and mechanism of essentially all aspects of DNA replication is the MCM2-7 complex, a hexameric ATPase believed to be the replicative helicase - the molecular motor that unwinds duplex DNA at the replication fork. As one of the few factors essential in both the initiation and elongation phases of DNA replication, defects in MCM activity can compromise the precision of DNA replication in multiple ways. In particular, displacement of the MCM2-7 complex from replication forks can potentially occur as it encounters various obstacles. Such displacement will lead to collapse of the replication fork, a defect that creates the types of genomic instability characteristic of cancer and birth defects. The following Aims build upon our previous work with the MCM2-7 complex: 1) Does defective DNA unwinding lead to replication fork collapse? Evidence suggests that MCM2-7 is essential for replication fork progression. However, our in vitro analysis indicates that MCM2-7 may be intrinsically inefficient at unwinding DNA, suggesting the need for additional factors in vivo. We will quantify the in vitro and in vivo ability of both wild type and mutant MCM2-7 complexes to unwind a variety of DNA substrates, assess the role that additional replication factors have on this activity, and use a genetic screen to identify novel factors that assist MCM2-7 fork progression. 2) Is a newly discovered discontinuity in the MCM2-7 complex required for initiation and helicase activation? Our in vitro analysis indicates that two MCM subunits form a reversible ATP-regulated "gate" in the toroidal structure. Using our MCM mutants defective for this activity in vitro, we propose to test the utility of this gate in vivo on the assembly of MCM2-7 onto DNA using chromatin immunoprecipitation, and activation of the MCM2-7 helicase by the replication specific kinase CDC7/DBF4. (3) What is the relationship between ATP binding, hydrolysis and DNA unwinding within the MCM2-7 complex? ATPases are abundant and perform diverse cellular functions, but considerable controversy exists as to how they couple ATP binding and hydrolysis to mechanical work. Unlike most ATPases, MCM2-7 has six distinct subunits that can be individually modified, making it ideal for studying the function these machines. We propose to mutate two specific structural motifs with predicted involvement in DNA binding and active site coordination - the pre- Sensor I insert and the Sensor II motif - then test the consequences on MCM2-7 activity using our established in vivo and in vitro functional assays.

Public Health Relevance

Defects in DNA replication lead to genomic instability, a condition that causes a predisposition to cancer, birth defects, and a variety of other human health issues. The MCM2-7 complex is central to both the regulation and precise replication of the genome. We propose to capitalize on our recent progress on understanding the mechanism of this complex to examine MCM2-7 regulation and its involvement in genomic instability.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM083985-05
Application #
8499356
Study Section
Molecular Genetics A Study Section (MGA)
Program Officer
Reddy, Michael K
Project Start
2009-07-01
Project End
2014-06-30
Budget Start
2013-07-01
Budget End
2014-06-30
Support Year
5
Fiscal Year
2013
Total Cost
$249,927
Indirect Cost
$75,428
Name
University of Pittsburgh
Department
Biology
Type
Schools of Arts and Sciences
DUNS #
004514360
City
Pittsburgh
State
PA
Country
United States
Zip Code
15213
Simon, Nicholas E; Schwacha, Anthony (2014) The Mcm2-7 replicative helicase: a promising chemotherapeutic target. Biomed Res Int 2014:549719
Bochman, Matthew L; Schwacha, Anthony (2010) The Saccharomyces cerevisiae Mcm6/2 and Mcm5/3 ATPase active sites contribute to the function of the putative Mcm2-7 'gate'. Nucleic Acids Res 38:6078-88