Modulation of DNA End Joining by Nuclear Proteins

Li Liang,Li Deng,Yanping Chen,Gloria C Li,Changshun Shao,Jay A Tischfield

doi:10.1074/jbc.m503776200

Abstract

DNA double strand breaks in mammalian cells are primarily repaired by homologous recombination and non-homologous end joining (NHEJ). NHEJ may either be error-free or mutagenic with deletions or insertions at the joint. Recent studies showed that DNA ends can also be joined via microhomologous sequences flanking the break point especially when proteins responsible for NHEJ, such as Ku, are absent. Microhomology-mediated end joining (MHEJ) is always accompanied by a deletion that spans one of the two homologous sequences and the intervening sequence, if any. In this study we evaluated several factors affecting the relative contribution of MHEJ to DNA end joining using nuclear extracts and DNA substrates containing 10-bp repeats at the ends. We found that the occurrence of MHEJ is determined by the relative abundance of nuclear proteins. At low DNA/protein ratios, an error-free end-joining mechanism predominated over MHEJ. As the DNA/protein ratio increased, MHEJ became predominant. We show that the nuclear proteins that contribute to the inhibition of the error-prone MHEJ include Ku and histone H1. Treatment of extracts with flap endonuclease 1 antiserum significantly reduced MHEJ. Addition of a 17-bp intervening sequence between the microhomologous sequences significantly reduced the efficiency of MHEJ. Thus, this cell-free assay provides a platform for evaluating factors modulating end joining.

Highlights

DNA double strand breaks (DSBs),1 the most serious form of DNA damage, can lead to cell death if not repaired
This microhomologydriven, error-prone end joining occurred in mammalian cells that are deficient for the other classical non-homologous end joining (NHEJ) proteins XRCC4 [7] and ligase IV [10]
Deletions generated via MHEJ are characteristic of single strand annealing (SSA), an error-prone homologous recombination repair pathway identified for yeast and mammalian cells [15,16,17,18]

Summary

Introduction

DNA double strand breaks (DSBs), the most serious form of DNA damage, can lead to cell death if not repaired. SSA is assumed to proceed by a series of steps including (a) the generation of single strands adjacent to the break and its extension to the repeated sequences such that the complementary strands can anneal to each other, (b) annealing of single strands at homology patches, (c) removal of unpaired flap strands by endoand/or exonucleases, (d) DNA polymerase-mediated filling in of gaps, and (e) sealing of remaining nicks by a DNA ligase [17] This process results in a deletion of one of the repeats and the sequence between the repeats and like MHEJ, is always mutagenic. Deficiency of proteins required for classical NHEJ has been shown to shift end joining to MHEJ in mammalian cells, a systematic study of the factors that modulate the choice between the two pathways has been lacking We postulated that both nuclear proteins and the DNA sequence context in which DSBs arise may dictate the route of their repair. The efficiency of MHEJ is affected by the size of repeats as well as the proximity of repeats

Methods

Results

Conclusion