Human cells respond to endogenous and exogenous stresses by using post translational modification as a fundamental tool to control protein functions. This regulation controls genome stability and DNA repair in ways that either promote or suppress carcinogenesis. We have evidence that protein acetylation specifically adjusts the fidelity of DNA replication and repair. One strand of the DNA genome is made discontinuously, whereby Okazaki fragments about 150 nucleotides long are synthesized and joined. Similar mechanisms, and mostly the same enzymes, are employed by the frequently-utilized base excision repair system. For replication, RNA/DNA primer of each fragment, made by the error-prone DNA polymerase ?, is raised into a flap by synthesis from the adjacent fragment. For repair, a damaged part of DNA is also made into a flap. In both cases the flap is removed by flap endonuclease (FEN1) and then adjacent segments are joined. Our previous reconstitution analyses showed that most replication/repair flaps were removed while short, so that a minimal-length synthesis patch was made before fragment joining. However, some flaps became long, and a large patch was replaced, requiring the additional function of the Dna2 nuclease for long flap cleavage. Why should two pathways, long and short flap creation and removal, have evolved? We have evidence that they represent a fundamental regulation process based on these observations: Acetylation of human FEN1 by the histone acetyltransferase p300 lowers nuclease activity. Acetylation of Dna2 nuclease by p300 greatly enhances cleavage activity. Acetylation stimulates DNA polymerases that displace the flaps. Together, these effects suggest a regulation in which longer flaps are created, properly processed by Dna2, but joined only after a long patch is replaced because of the lower FEN1 activity. Such regulation would cause replication/repair proteins to replace a longer patch, assuring Okazaki primer removal in replication and complete damage removal in repair. The regulation would balance fidelity with efficiency. Preliminary evidence in yeast having an enhanced error-prone DNA polymerase ?, showing that deletion of a major protein acetylase decreases fidelity of DNA replication, supports this hypothesis. Our proposal has two key components: One, the enzymatic changes caused by acetylation of replication/repair proteins will be defined for individual proteins, interacting protein partners, and reconstitutions of the replication and repair pathways. Results should reveal whether acetylation changes protein function consistent with the original hypotheses, or suggest alternative explanations. Two, genetic and cell biological approaches will be applied to determine the effects of replication/repair protein acetylation in the cell. We ill specifically establish whether acetylation is used to regulate patch replacement length in replication/repair, and whether that regulation alters the fidelity of DNA synthesis.

Public Health Relevance

Endogenous and exogenous stresses to the human cell regulate the rate of human aging, the preservation state of genetic information, and the probabilities of carcinogenesis. Recent evidence suggests that these environmental factors alter DNA replication and repair to be either more efficient or more accurate. We are investigating this regulation process with the goal of intervening to augment natural protection of genetic information for better human health.

Agency
National Institute of Health (NIH)
Institute
National Cancer Institute (NCI)
Type
Exploratory/Developmental Grants (R21)
Project #
5R21CA176249-02
Application #
8788391
Study Section
Special Emphasis Panel (ZCA1-RPRB-B (O1))
Program Officer
Witkin, Keren L
Project Start
2014-04-01
Project End
2016-03-31
Budget Start
2015-04-01
Budget End
2016-03-31
Support Year
2
Fiscal Year
2015
Total Cost
$200,318
Indirect Cost
$69,818
Name
University of Rochester
Department
Microbiology/Immun/Virology
Type
Schools of Dentistry
DUNS #
041294109
City
Rochester
State
NY
Country
United States
Zip Code
14627
Piekna-Przybylska, Dorota; Bambara, Robert A; Balakrishnan, Lata (2016) Acetylation regulates DNA repair mechanisms in human cells. Cell Cycle 15:1506-17