1 MCB 502A-2015. Lecture #14. Recombinational Repair. Classification of chromosomal lesions: types of "failure-by-design" Daughter-strand gaps and collapsed replication forks are replication-dependent chromosomal lesions and as such look quite different from direct chromosomal lesions — double-strand breaks and interstrand crosslinks. However, we can group these four lesions into two major classes. When a cell tries to repair a crosslink, it first excises one-strand portion of it (in the so-called "unhooking reaction"), but the repair cannot be complete, because the second strand is also damaged and cannot be used as a template to resynthesize the excised DNA strand. Therefore, interstrand crosslink is converted into a blocked (unfillable) single-strand gap and, in this quality, resembles daughter-strand gaps. In contrast to a direct double-strand break, replication fork collapse generates one double-strand end. However, a double-strand break can be viewed as a special case of replication fork collapse that generates two double-strand ends. Indeed, if there is a nick in the terminus of a theta-replicating DNA, and the replication forks are converging on this nick from both directions, the result will be one intact chromosome and one chromosome with a double-strand break in place of the nick. Thus, we can group interstrand crosslinks and DSGs into the group of persistent single-strand gaps (one strand interrupted), while DSBs and RFCs into the group of double-strand ends (two strands interrupted). Finally, the replication-independent chromosomal lesions without strand interruption are a chromosomal dimer and a DNA knot, whereas their replication-dependent counterparts are a locked replication fork and catenated sister chromosomes. Thus, we can make an attempt at classifying chromosomal lesions using, on the one hand, the number of DNA strands interrupted, while on the other hand, based on whether the lesions is direct or replication-dependent. As was already mentioned on a couple of occasions, besides the defining quality of interferance with the chromosome cycle, all chromosomal lesions have another property in common that sets them apart from one-strand DNA lesions/modifications: chromosomal lesions cannot be repaired by excision repair pathways. This is because excision repair depends on the intactness of the second DNA strand across the lesion, while all chromosomal lesions either affect both DNA strands opposite each other, so there is no intact template for repair, or have no damaged DNA at all. In this respect, chromosomal lesions look like "natural design vulnerabilities" (similar to zero-day vulnerabilities in programming), providing insidious ways of killing cells of any type via irreversible inactivation of their genetic material. Chromosomal lesions are repaired by Rec-dependent processes Are chromosomal lesions repairable at all? And if yes, what could be the mechanisms of their repair? Whenever we are clueless about any Life phenomenon, we reach for Genetics to get the initial answers. In this case we could isolate mutants that are sensitive to DNA-damaging treatments and then, among these mutants using our physical read-outs, find those that are unable to repair chromosomal lesions. For example, we have already isolated mutants, sensitive to UV-light. Moreover, we even did the epistatic analysis, constructing double mutants and checking their phenotypes against single mutants, to group all the UV-sensitive mutants into three major categories: 1) mutants deficient in photoreactivation (phr); 2) mutants deficient in the nucleotide excision repair (uvr); 3) rec mutants, called so because they are deficient in a complex process of homologous recombination: Group Primary Defect Removal of PD DSB / DSG repair2 phr photoreactivation +, by NER normal uvr nucleotide excision repair ±, by PhL and light normal rec homologous recombination ++, by NER and PhL defective When researchers checked how these three groups of mutants repair chromosomal lesions, they found that the phr and uvr mutants repair DSBs and DSGs no problem, but some rec mutants were completely deficient in chromosomal repair. For example, alkaline sucrose gradients reveal lower molecular weight of the newly-synthesized DNA in uvr mutants after UV-irradiation, indicating formation of daughter-strand gaps. However, further incubation in the growth medium leads to this DNA gradually becoming high molecular weight, indicating repair of daughter-strand gaps and showing that this return to normalcy is independent of the nucleotide excision repair. Importantly, the lower molecular weight nascent DNA stays LMW in the recA mutants, indicating no repair. Since the rec mutants were also deficient in homologous recombination, the natural idea was that homologous recombination can be used to repair chromosomal lesions, caused by DNA strand damage. Homologous recombination: exchange by resolution of Holliday junctions What is homologous recombination? If two proteins or two DNAs are mostly identical in their sequences, they are called “homologous” (“agreeing in general content”). In the long chromosomes, even if they are sisters, it is hard to maintain 100% identity, so "homology" is a more encompassing and biologically-relevant term. Homologous recombination is exchange between two chromosomes (or within a chromosome) in the aligned regions of sequence identity. A generic mechanism of homologous recombination begins with separation and exchange of strands between two homologous DNA duplexes. Since the duplexes are identical, their strands, when aligned reciprocally, can pair to form two hybrid duplexes (with the help of topoisomerases, of course) in the so-called “recombination intermediate”. The places in the recombination intermediate where the two original duplexes end and the hybrid duplexes begin are called Holliday junctions, after Robin Holliday, who first proposed their existence and explained their significance. Holliday junctions can be seen in EM in recombining DNAs. But how to finish the recombination reaction, how to proceed from the recombination intermediate to the products? Holliday proposed that DNA junctions are “resolved” by single-strand cleavages in either one or the other pair of opposite strands. The two alternative ways to resolve a Holliday junction can be better imagined if we isomerize the junction by rotating two adjacent
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