Substrate preference of Gen endonucleases highlights the importance of branched structures as DNA damage repair intermediates
Stephanie P. BellendirDanielle J. RognstadLydia P. MorrisGrzegorz ZapotocznyWilliam G. WaltonMatthew R. RedinboDale A. RamsdenJeff SekelskyDorothy A. Erie
0
Citation
0
Reference
10
Related Paper
Reactivity
Cite
Citations (4)
Cite
Citations (10)
Cell damage
Cell fate determination
Robustness
CHEK1
Cite
Citations (49)
CHEK1
Cite
Citations (8)
MCL-1, a pro-survival member of the BCL-2 family, was previously shown to have functions in ATR-dependent Chk1 phosphorylation following DNA damage. To further delineate these functions, we explored possible differences in DNA damage response caused by lack of MCL-1 in mouse embryo fibroblasts (MEFs). As expected, Mcl-1-/- MEFs had delayed Chk1 phosphorylation following etoposide treatment, compared to wild type MEFs. However, their response to hydroxyurea, which causes a G1/S checkpoint response, was not significantly different. In addition, appearance of g-H2AX was delayed in the Mcl-1-/- MEFs treated with etoposide. We next investigated whether MCL-1 is present, together with other DNA damage response proteins, at the sites of DNA damage. Immunoprecipitation of etoposide-treated extracts with anti-MCL-1 antibody showed association of MCL-1 with g-H2AX as well as NBS1. Immunofluorescent staining for MCL-1 further showed increased co-staining of MCL-1 and NBS1 following DNA damage. By using a system that creates DNA double strand breaks at specific sites in the genome, we demonstrated that MCL-1 is recruited directly adjacent to the sites of damage. Finally, in a direct demonstration of the importance of MCL-1 in allowing proper repair of DNA damage, we found that treatment for two brief exposures to etoposide over several days, which mimics the clinical situation of etoposide use, resulted in many more chromosomal abnormalities in the MEFs that lacked MCL-1. Together, these data indicate an important role for MCL-1 in coordinating DNA damage mediated checkpoint response, and have broad implications for the importance of MCL-1 in maintenance of genome integrity.
Immunoprecipitation
Cite
Citations (59)
Hypoxanthine
Xanthine
Cite
Citations (13)
The water-soluble steroid (I) acts as a catalyst for the hydrolysis of active esters, with a definite specificity for esters of 3-arylpropionic acids, the ester of 3-(3-phenanthryl)propionic acid being the most rapidly hydrolysed substrate found to date; it is concluded that imidazole-catalysed hydrolysis of the esters is facilitated by hydrophobic interactions between substrate and catalyst.
Imidazole
Ester hydrolysis
Cite
Citations (4)
The BRCA1 gene product and its stoichiometric binding partner, BARD1, play a vital role in the cellular response to DNA damage. However, how they acquire specific biochemical functions after DNA damage is poorly understood. Following exposure to genotoxic stress, DNA damage-specific interactions were observed between BRCA1/BARD1 and the DNA damage-response proteins, TopBP1 and Mre11/Rad50/NBS1. Two distinct DNA damage-dependent super complexes emerged; their activation was dependent, in part, on the actions of specific checkpoint kinases, and each super complex contributed to a distinctive aspect of the DNA damage response. The results support a new, multifactorial model that describes how genotoxic stress enables BRCA1 to execute a diverse set of DNA damage-response functions.
Rad50
CHEK1
Cite
Citations (305)
Michaelis–Menten kinetics
Enzyme Kinetics
Cite
Citations (20)