Hypothesis: Homologous Recombination Depends on Parallel Search

Abstract

Having a double stranded break (DSB) of chromosomal DNA is a serious condition for any cell. In all kingdoms of life cells achieve error free chromosome repair after DSB by using a reference sequence in a sister chromatid or a homologous chromosome through the process of homologous recombination (HR). The molecular mechanism of HR has been worked out in great detail [1], since HR deficiency is a major source of cancer [2]. However, the mystery of how a broken sequence can find its homologous reference sufficiently fast is unsolved [3].The problem is that the homologous reference sequence can be anywhere in the cell [4]. Thus, HR has to depend on probing each conceivable chromosome position although this requires unwinding of the double helix and base pairing with the broken chromosome sequence. Even considering recent in vitro measurements revealing rapid sampling with short oligos [5] it would take more than a year to unwind and compare all sequences in a mammalian genome and weeks for yeast genome. If we add the diffusive and topological constraints of moving and aligning the broken chromosome in a dense nucleus, the time scales involved become astronomical, especially considering that the broken ends must also be held together [6]. Still the cell manages to find the homologous sequence in hours [7]. Something fundamental is clearly missing in our understanding of homologous recombination.If it takes too long to solve a problem sequentially it can often be parallelized. Thus, I suggest that the cell makes many short copies of the sequences flanking the DBS and use these to search for the homologous sequence in parallel. This strategy not only parallelizes the search process but also eliminates the need to move the broken chromosome around in a time consuming manner. Although the necessary molecular components to implement such a strategy have already have been characterized, the transformative impact of this strategy on search speed has not been discussed.For example, in Arabidopsis and in human cells, 21 nt RNAs are made, corresponding to the sequences flanking the DBS [8]. These so called diRNA bind to Ago2, which from separate studies, e.g. ref [9], are seen targeting chromosomal promoters or nascent transcripts when programmed by homologous 21 nt microRNA. Thus, the Ago2 diRNA complexes are ideal candidates for searching the genome for sequences homologous to the broken sequence. Ago2 deletions are further known to be deficient in HR and Ago2 is directly binding to the critical HR protein Rad51 even in the absence of the diRNA [10].An RNA corresponding to the diRNA has not been described in bacteria, but the many short ssDNA resection products that are made by the recBCD or addAB systems while processing the DSB ends [11], could play the same role. This would also explain why ssDNA degradation products have lengths of tens of nucleotides [12]. In fact, short pieces of ssDNA bind RecA in vitro and the complex also search for specific sequences in dsDNA [13], but it is not known whether the resection product...

DNA Repair: The Search for Homology

“…To search each of 10 7 unique alignments would leave only 0.004 sec per inquiry. To explain and overcome what seems like an impossible barrier to successful repair, several possible solutions have been proposed, including that homologous sequences are pre-aligned before a DSB occurs or that some diffusible copies of the template are used [60, 61]. But in fact, the search problem is not as formidable as it first seems.…”

The Good and Bad of RNA:DNA Hybrids in Double-Strand Break Repair

“…There have been a number of observations suggesting a role for transcription and non-coding RNAs in response to DSBs and some suggestions about how this might facilitate the repair process, such as opening of chromatin via transcription or with the RNAs as platforms for repair proteins, chromatin remodelers, or histone modifiers (Gao et al, 2014;Wang and Goldstein, 2016;Wei et al, 2012). Other, more direct roles for RNA in DSB repair include the RNA serving as repair templates (Keskin et al, 2014) or facilitating the homology search process (Elf, 2016). Ohle et al (2016) find a seeming paradox: RNase H activity is necessary for efficient homologous recombination, yet in excess, it prevents efficient homologous recombination in Schizosaccharomyces pombe.…”

“…If a single DNA end must search the entire genome for potential homology and sample all possibilities, it would take considerable time to unpack and query the entire yeast genome. Elf proposes that the search is ''paralellized'' so that multiple copies of the same sequence are searching the genome and that either RNA or DNA might be used (Elf, 2016). If RNA copies were generated to facilitate the search, they would eventually need to be removed to allow the proper DNA end to access the homology.…”