The faithful propagation of genetic material is essential to life. A central prerequisite to the copying of DNA is the dedicated assembly of replicative machineries at proper sites on the chromosome. This replication """"""""initiation"""""""" event depends on multiple components, including: 1) initiators, ATPases that bind origins and recruit other proteins to the replication start site, 2) helicase-loaders, which deposit replicative helicases onto DNA, and 3) primases, enzymes that create short oligonucleotides for extension by DNA polymerases. A long-term objective of our research has been to understand the molecular structure/function relationships that govern the initiation of DNA replication. Many of the proteins responsible for initiation have been identified, and a preliminary framework for their action is in place. However, there remains a host of outstanding questions regarding how these factors act individually and cooperatively to construct a competent replication fork. Our prior efforts in this area have generated new mechanistic models for initiator action, helicase loading, and priming that we are now in an ideal position to test. Using a combination of structural, biochemical, and biophysical methods we aim to: 1) Define how ATP controls DNA binding and remodeling by prokaryotic initiators, 2) Determine how ATP regulates bacterial helicase-loader assembly and function, and 3) Establish how the bacterial primase synthesizes primers and is inhibited by disparate types of small-molecule agents, including the stringent response regulator, (p)ppGpp. Together, these efforts will impact a number of scientific fronts, from understanding how origins are engaged and restructured to promote replisome construction, to defining how chemical energy and small-molecule inhibitors control the function of dynamic macromolecular machines. Knowledge of these processes is necessary for understanding how cells ultimately avoid initiation errors linked to genomic instabilities, transformation, and neoplasia.

Public Health Relevance

The faithful propagation of genetic material is essential to life. The goal of our research is understand the structure/function relationships that govern the initiation of DNA replication at a molecular level. Knowledge of these events is necessary for understanding how cells ultimately avoid errors linked to genomic instabilities and cancer onset.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM071747-08
Application #
8267665
Study Section
Special Emphasis Panel (ZRG1-GGG-H (02))
Program Officer
Preusch, Peter C
Project Start
2005-05-01
Project End
2013-06-30
Budget Start
2012-05-01
Budget End
2013-06-30
Support Year
8
Fiscal Year
2012
Total Cost
$292,005
Indirect Cost
$88,654
Name
University of California Berkeley
Department
Biochemistry
Type
Schools of Arts and Sciences
DUNS #
124726725
City
Berkeley
State
CA
Country
United States
Zip Code
94704
Lawson, Michael R; Dyer, Kevin; Berger, James M (2016) Ligand-induced and small-molecule control of substrate loading in a hexameric helicase. Proc Natl Acad Sci U S A 113:13714-13719
Hood, Iris V; Berger, James M (2016) Viral hijacking of a replicative helicase loader and its implications for helicase loading control and phage replication. Elife 5:
Thomsen, Nathan D; Lawson, Michael R; Witkowsky, Lea B et al. (2016) Molecular mechanisms of substrate-controlled ring dynamics and substepping in a nucleic acid-dependent hexameric motor. Proc Natl Acad Sci U S A 113:E7691-E7700
Hauk, Glenn; Berger, James M (2016) The role of ATP-dependent machines in regulating genome topology. Curr Opin Struct Biol 36:85-96
Arias-Palomo, Ernesto; Berger, James M (2015) An Atypical AAA+ ATPase Assembly Controls Efficient Transposition through DNA Remodeling and Transposase Recruitment. Cell 162:860-71
Petojevic, Tatjana; Pesavento, James J; Costa, Alessandro et al. (2015) Cdc45 (cell division cycle protein 45) guards the gate of the Eukaryote Replisome helicase stabilizing leading strand engagement. Proc Natl Acad Sci U S A 112:E249-58
Bleichert, Franziska; Botchan, Michael R; Berger, James M (2015) Crystal structure of the eukaryotic origin recognition complex. Nature 519:321-6
O'Shea, Valerie L; Berger, James M (2014) Loading strategies of ring-shaped nucleic acid translocases and helicases. Curr Opin Struct Biol 25:16-24
Costa, Alessandro; Renault, Ludovic; Swuec, Paolo et al. (2014) DNA binding polarity, dimerization, and ATPase ring remodeling in the CMG helicase of the eukaryotic replisome. Elife 3:e03273
Duderstadt, Karl E; Berger, James M (2013) A structural framework for replication origin opening by AAA+ initiation factors. Curr Opin Struct Biol 23:144-53

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