DNA double stranded-breaks (DSBs) are thought to be the most dangerous DNA lesions that threaten genomic integrity. In vertebrates, the classical non-homologous end joining pathway (c-NHEJ) joins most DSBs in all cell types and at all stages of the cell cycle. Not surprisingly, intact c-NHEJ is essential for human life. Disruption o c-NHEJ in somatic cells results in genomic instability and is strongly associated with tumorigenesis. Paradoxically, strategies for disrupting c-NHEJ, may have several potentially important therapeutic applications. Thus, a solid understanding of this pathway is important to human health. Although c-NHEJ has been studied for decades, more basic research is warranted because our knowledge of c-NHEJ is not only incomplete, but may also be inaccurate. The current research focuses on three areas. The first will ascertain mechanistic details of how phosphorylations mediated by the DNA dependent protein kinase (DNA-PK) regulate DNA repair. DNA-PK is a large protein kinase complex that initiates DNA repair. It targets (by protein phosphorylation) all of the c-NHEJ factors including itself. This autophosphorylation is essential for repair and is functionally complex. The second area of research will focus on emerging data demonstrating how DNA-PK affects other DNA repair pathways. c-NHEJ should not join certain types of DNA damage;one example of damage that c-NHEJ should preferably avoid is the damage that results during DNA replication when a replication fork collapses.
The second aim will determine whether DNA-PK promotes cell survival during replication stress at the expense of genome integrity.
The third aim follows up on recent novel findings showing how XRCC4 and XLF [two factors previously ascribed to c-NHEJ's ligation complex, and thus factors that function late in c-NHEJ] form filamentous structures that bridge DNA in vitro, and that may function in living cells to stabilize DNA ends prior to repair. This suggests an additional, early (in c-NHEJ) role for XRCC4/XLF filaments. This would be a significant shift from current dogma.

Public Health Relevance

DNA (the blue print for all life) is very sensitive and can be damaged in many ways;the pathway that repairs double stranded-breaks is essential to human life. Disruption of this pathway prior to birth results in Severe Combined Immunodeficiency (SCID), and in most cases, severe brain defects;whereas disruption of this pathway after birth can lead to cancer. The current research will ascertain mechanistic details of this DNA repair pathway;this basic research is warranted because our knowledge of this type of DNA repair is not only incomplete, but may also be inaccurate.

Agency
National Institute of Health (NIH)
Institute
National Institute of Allergy and Infectious Diseases (NIAID)
Type
Research Project (R01)
Project #
2R01AI048758-14
Application #
8435667
Study Section
Cellular and Molecular Immunology - A Study Section (CMIA)
Program Officer
Nasseri, M Faraz
Project Start
2000-03-15
Project End
2017-04-30
Budget Start
2013-05-15
Budget End
2014-04-30
Support Year
14
Fiscal Year
2013
Total Cost
$355,242
Indirect Cost
$120,242
Name
Michigan State University
Department
Pathology
Type
Schools of Veterinary Medicine
DUNS #
193247145
City
East Lansing
State
MI
Country
United States
Zip Code
48824
Neal, Jessica A; Xu, Yao; Abe, Masumi et al. (2016) Restoration of ATM Expression in DNA-PKcs-Deficient Cells Inhibits Signal End Joining. J Immunol 196:3032-42
Meek, Katheryn; Xu, Yao; Bailie, Caleb et al. (2016) The ATM Kinase Restrains Joining of Both VDJ Signal and Coding Ends. J Immunol 197:3165-3174
Masani, Shahnaz; Han, Li; Meek, Katheryn et al. (2016) Redundant function of DNA ligase 1 and 3 in alternative end-joining during immunoglobulin class switch recombination. Proc Natl Acad Sci U S A 113:1261-6
Ying, Songmin; Chen, Zhihui; Medhurst, Annette L et al. (2016) DNA-PKcs and PARP1 Bind to Unresected Stalled DNA Replication Forks Where They Recruit XRCC1 to Mediate Repair. Cancer Res 76:1078-88
Roy, Sunetra; de Melo, Abinadabe J; Xu, Yao et al. (2015) XRCC4/XLF Interaction Is Variably Required for DNA Repair and Is Not Required for Ligase IV Stimulation. Mol Cell Biol 35:3017-28
Douglas, Pauline; Ye, Ruiqiong; Trinkle-Mulcahy, Laura et al. (2014) Polo-like kinase 1 (PLK1) and protein phosphatase 6 (PP6) regulate DNA-dependent protein kinase catalytic subunit (DNA-PKcs) phosphorylation in mitosis. Biosci Rep 34:
Neal, Jessica A; Sugiman-Marangos, Seiji; VanderVere-Carozza, Pamela et al. (2014) Unraveling the complexities of DNA-dependent protein kinase autophosphorylation. Mol Cell Biol 34:2162-75
Mahaney, Brandi L; Hammel, Michal; Meek, Katheryn et al. (2013) XRCC4 and XLF form long helical protein filaments suitable for DNA end protection and alignment to facilitate DNA double strand break repair. Biochem Cell Biol 91:31-41
Müller, Tina A; Yu, Kefei; Hausinger, Robert P et al. (2013) ALKBH1 is dispensable for abasic site cleavage during base excision repair and class switch recombination. PLoS One 8:e67403
Woodbine, Lisa; Neal, Jessica A; Sasi, Nanda-Kumar et al. (2013) PRKDC mutations in a SCID patient with profound neurological abnormalities. J Clin Invest 123:2969-80

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