Here we report the first analysis of NHEJ and HR in normal human cells. We employ sensitive fluorescent reporter assays that allow for a direct comparison of the efficiencies of NHEJ and HR events upon induction of chromosomal DSBs with a rare-cutting endonuclease. Fluorescent assays allow for scoring DSB repair events in thousands of cells, and are highly quantitative. Furthermore, rather than relying on a single genomic locus we analyzed DSB repair at multiple chromosomal locations. By doing so we demonstrate that the efficiency of NHEJ and HR is strongly affected by chromosomal position. Nevertheless, when NHEJ and HR are compared across multiple integration sites, NHEJ is more efficient than HR. In unsynchronized proliferating cell populations, NHEJ of compatible DNA ends is twice as efficient as NHEJ of incompatible DNA ends, and NHEJ of incompatible DNA ends is three times more efficient than HR. Since the majority of randomly occurring DSBs have DNA ends that require processing prior to ligation we conclude that in proliferating cells NHEJ repairs 75% of DSBs while HR repairs the remaining 25%. This ratio is similar to the one reported for mouse ES cells [9
], however in that study the DSB repair pathways were distinguished by analyzing repair products by Southern blot and only 42 colonies were examined. An overall 3:1 ratio between NHEJ and HR may be a general phenomenon for mammalian cells, although for every individual break it is strongly affected by chromosomal location and the type of DNA ends. It should be noted that the observed frequencies refer to actively proliferating cells. In G1-arrested quiescent or differentiated cells the frequency of HR is likely to be much lower [4
]. The ratio between NHEJ and HR varies greatly across phylogenetic groups. Yeast rely heavily on HR while in mammals and plants NHEJ is the preferred pathway [11
]. The choice may be dictated by genome composition [12
]. In large repetitive genomes of plants and animals overly efficient HR may lead to deleterious genomic rearrangements, such that NHEJ may be a safer choice. The trade-off is the accumulation of small mutations resulting from error-prone NHEJ. Thus, mammalian cells may avoid large genomic rearrangements, but instead accumulate deletions and insertions that contribute to aging and tumorigenesis.
We performed comparison of the kinetics of NHEJ and HR. Our assay allows for rapid and transient expression of I-SceI and an almost instantaneous expression and detection of GFP. We show that NHEJ is much faster than HR. NHEJ can be completed in approximately 30min, while HR takes 7h or more. The kinetics of HR has been analyzed in detail in yeast by directly monitoring recombination products by Southern blot or PCR. The process of recombination was slow(for a fast growing organism like Saccharomyces
) and took from 1h for mating type switching [15
] to up to 3h for repair of a substrate containing LACZ gene duplication [16
]. The kinetics of DSB repair in mammalian cells have previously been examined by pulse field gel electrophoresis and analysis of gamma-H2AX foci [17
]. Considering that this technique monitors primarily NHEJ events, the kinetics of NHEJ that we observed is consistent with the earlier studies where the majority of DSBs were repaired within 90 min [17
]. The in situ
studies of Rad51 recruitment to radiation-induced foci showed that Rad51 is recruited within 1h of DSB induction and leaves after 5h [21
]. It is likely that departure of Rad51 does not manifest the completion of repair, as any remaining gaps, heteroduplexes, and nicks must be filled-in, repaired, and ligated. Therefore, the 7h time course for completion of HR that we observe corresponds well with in situ
studies. It is remarkable that HR takes such a long time to complete. What factors determine the choice between NHEJ and HR remain amystery and a subject of intense studies. Our finding that NHEJ is a much faster process than HR offers an explanation of the higher efficiency of NHEJ in mammalian cells.