MCB 502A-2014. Lecture #14. Recombinational Repair.Chromosome cycle-dependent chromosomal lesionsDouble-strand breaks and interstrand crosslinks are classified as direct chromosomal lesions, because they do not require any other endogenous (cellular) processes for their formation. However, they do assume that cells encounter unusual exogenous interference in the form of high-energy ionizing radiation or cross-linking agents. For this reason, direct chromosomal lesions represent a small minority of all chromosomal lesions that could form in growing cell. The bulk of chromosome lesions in cycling cells are due to transitions of the chromosome cycle on non-intact DNA. Replication-dependent chromosomal lesions are induced when replication forks run into unrepaired one-strand lesions in template DNA. For example, replicating DNA in uvr mutant cells, which were irradiated with UV, is analyzed in sucrose gradients, which can distinguish chromosome-size DNA pieces from smaller pieces. Pyrimidine dimers in uvr mutant cells are not removed, and so replication forks in UV-irradiated uvrA mutants will encounter PDson the regular basis. If one runs pre-labeled (labeled before UV) chromosomal DNA from these cells through alkaline sucrose gradient, one will see no change from unirradiated control cells. However, if one now labels DNA in these cells for 10 minutes after UV irradiation, so that only the newly-replicated DNA is labeled, one will see that the label in unirradiated cells accumulates in HMW species, as first described by Okazaki (because the labeling time is so long), but in the irradiated cells, the label accumulates in lower-molecular weight species, and the heavier the irradiation, the lower the molecular weight of the newly-synthesized DNA becomes. This result suggests that, when replicating DNA strands encounter PDs, they have to go around them, leaving behind single-strand interruptions, whose number roughly corresponds to the number of the original PDs, encountered by the replication forks. Actually, these are not just interruptions or even short gaps opposite PDs — the size of the gaps is approximately half the length of Okazaki fragments — from 500 to 1,000 nucleotides. They are called daughter-strand gaps, because they are generated only in the newly-synthesized DNA strands. It is proposed that when a replication fork encounters an unrepaired pyrimidine dimer, the DNA polymerase copying the affected strand is stalled, but the replication fork continues past the lesion, recruiting another polymerase and reinitiating downstream. Such amaneuver leaves a portion of the newly-synthesized strand unreplicated, — thus, a daughter-strand gap. The daughter-strand gap is a chromosomal lesion, because it precludes subsequent replication of the affected chromosome. Indeed, both strands of the duplex with a daughter-strandgap now cannot serve as templates for subsequent replication: one has the gap, the other still has the original PD. When a daughter-strand gap happens to form in excision-repair proficient cells, there is yet another unpleasant outcome: since the PD is now in a single-strand DNA, it cannot beexcised by NER, which works only in duplex DNA. 1If we analyze a replicating chromosome from UV-irradiated WT cells (nucleotide-excision repair is operational in this case), we will see, in contrast to the uvr mutant cells, that themolecular weight of the pulse-labeled DNA strands in alkaline sucrose gradients approaches the one of the intact chromosomal DNA, suggesting a greatly reduced formation of daughter-strand gaps. This is expected, because in WT cells, PDs are rapidly removed by nucleotide-excision repair. However, if we run a control, by testing the overall chromosome status in neutral sucrose gradients, then we will see that the MW of duplex DNA is actually decreased, suggesting that thechromosome fragments (forms occasional double-strand breaks) in these conditions. If true, these breaks should also affect the old DNA strands (labeled before UV-irradiation). In fact, they do: a fraction of the old DNA strands (expected to be in duplex with strands synthesized after UV) is also broken. Remarkably, this chromosomal fragmentation is blocked by either blocking DNA replication or by inactivating excision repair! In other words, the chromosome is fragmented only in cells that are 1) in the process of chromosome replication and 2) are at the same time removing PDs by excision repair. What is thought to happen in these cells is the replication fork collision with excision intermediates, — various kinds of single-strand interruptions or short gaps. According to this scenario, when a replication fork runs into such an interruption in one of the strands of the template DNA, the replication fork falls apart — we say, it “collapses”. Eventually, the single-strand interruption that caused replication fork collapse will be repaired by the combined action of DNA pol I and DNA ligase, but the double-strand end will be open to degradation by exonucleases. You may think that there is nothing really wrong if the chromosome, instead of theta-replication will be sigma-replicating, using the surviving replication fork of the burst replication bubble — after all, new replication bubbles can still be initiated in the circular part ofthe chromosome. However, these new initiations can only lengthen the ever-growing linear tail, and such a sigma-replicating chromosome cannot produce a circular chromosome to segregate, — this is the so-called “sigma-replication trap”, leading to a moribund cell lineage. Chromosome lesions (at least double-strand breaks) also form during other stages of the chromosome cycle — for example, during chromosome compaction/condensation or segregation,— if these transactions operate on damaged DNA undergoing excision repair. In general, chromosome-cycle-dependent lesions by far outnumber direct chromosomal lesions because they are formed as a result of chromosome cycle (either a regular or almost continuous event) operating on DNA with one-strand lesions, that probably happen multiple times per generation, especially in cells of higher eukaryotes. 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 breaksand interstrand crosslinks. However, we can group
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