Abstract
Genomic instability is a known precursor to cancer and aging. The RecQ helicases are a highly conserved family of DNA-unwinding enzymes that play key roles in maintaining genome stability in all living organisms. Human RecQ homologs include RECQ1, BLM, WRN, RECQ4, and RECQ5β, three of which have been linked to diseases with elevated risk of cancer and growth defects (Bloom Syndrome and Rothmund-Thomson Syndrome) or premature aging (Werner Syndrome). RECQ1, the first RecQ helicase discovered and the most abundant in human cells, is the least well understood of the five human RecQ homologs. We have previously described that knockout of RECQ1 in mice or knockdown of its expression in human cells results in elevated frequency of spontaneous sister chromatid exchanges, chromosomal instability, increased load of DNA damage and heightened sensitivity to ionizing radiation. We have now obtained evidence implicating RECQ1 in the nonhomologous end-joining pathway of DNA double-strand break repair. We show that RECQ1 interacts directly with the Ku70/80 subunit of the DNA-PK complex, and depletion of RECQ1 results in reduced end-joining in cell free extracts. In vitro, RECQ1 binds and unwinds the Ku70/80-bound partial duplex DNA substrate efficiently. Linear DNA is co-bound by RECQ1 and Ku70/80, and DNA binding by Ku70/80 is modulated by RECQ1. Collectively, these results provide the first evidence for an interaction of RECQ1 with Ku70/80 and a role of the human RecQ helicase in double-strand break repair through nonhomologous end-joining.
Highlights
A DNA double-strand break (DSB) is detrimental to genome integrity [1]
Our previous observations that RECQ1 deficiency leads to cellular sensitivity to ionizing radiation or hydrogen peroxide [24,25,26] that potentially leads to DSBs raised the possibility that RECQ1 plays a direct role in DSB repair
Given the critical importance of the Ku70/80 heterodimer in nonhomologous endjoining (NHEJ) repair of DSBs, we characterized the putative interaction of RECQ1 with the Ku proteins
Summary
A DNA double-strand break (DSB) is detrimental to genome integrity [1]. DSBs are generated naturally in cells during programmed genome rearrangements [2,3], and as a consequence of problems in DNA metabolism, such as replication fork collapse or DNA damage induced by extrinsic mutagens including radiations [4]. Accurate repair of DSBs is indispensable to genome homeostasis and cell survival. Homologous recombination (HR) and nonhomologous endjoining (NHEJ) are mechanistically distinct DNA repair pathways that contribute substantially to DSB repair in mammalian cells [7]. HR utilizes an unbroken, homologous sequence as a template for repair of a DSB, thereby ensuring that any genetic information disrupted or lost at the site of the break is regained accurately. NHEJ is a second prominent pathway for DSB repair in which broken ends are healed without the requirement for significant sequence homology [9]. Mammalian cells preferentially utilize NHEJ for DSB repair throughout the cell cycle and exclusively during G1 to early S phase when the homologous template is unavailable for HR [10]. A number of proteins including the MRE11/RAD50/NBS1 (MRN) complex, BRCA1 and PARP-1 are shown to modulate both pathways but it is yet unclear how the choice is made between the HR and NHEJ pathways for the repair of a DSB [14]
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