SUMMARY Accurate and complete replication of the genome in every cell division is a prerequisite of genomic stability. Thus, both prokaryotic and eukaryotic replication forks are extremely precise and robust molecular machines that have evolved to be up to the task. However, it has recently become clear that the replication fork is more of a hurdler than a runner: it must overcome various obstacles present on its way. Such obstacles can be called natural impediments to DNA replication, as opposed to external and genetic factors. Natural impediments to DNA replication are particular DNA binding proteins, unusual secondary structures in DNA, and transcription complexes that occasionally (in eukaryotes) or constantly (in prokaryotes) operate on replicating templates. This review describes the mechanisms and consequences of replication stalling at various natural impediments, with an emphasis on the role of replication stalling in genomic instability.
While collisions between replication and transcription in bacteria are deemed inevitable, the fine details of the interplay between the two machineries are poorly understood. In this study, we evaluate the effects of transcription on the replication fork progression in vivo, by using electrophoresis analysis of replication intermediates. Studying Escherichia coli plasmids, which carry constitutive or inducible promoters in different orientations relative to the replication origin, we show that the mutual orientation of the two processes determines their mode of interaction. Replication elongation appears not to be affected by transcription proceeding in the codirectional orientation. Head-on transcription, by contrast, leads to severe inhibition of the replication fork progression. Furthermore, we evaluate the mechanism of this inhibition by limiting the area of direct contact between the two machineries. We observe that replication pausing zones coincide exactly with transcribed DNA segments. We conclude, therefore, that the replication fork is most likely attenuated upon direct physical interaction with the head-on transcription machinery.In bacteria, DNA replication and transcription continue throughout the life cycle. The speed of the replication fork progression in Escherichia coli is ϳ1,000 bp per s (16), while the elongation rate for RNA polymerase is just 50 nucleotides (nt) per s (15); i.e., replication is approximately 20-fold faster than transcription. Since the two processes proceed simultaneously, frequent collisions between replication and transcription seem unavoidable (3, 38). Given that both processes are polar, the collisions can occur either head-on or codirectionally (Fig. 1). In the case of head-on collisions, the front edge of RNA polymerase meets the hexameric DNA helicase DnaB that moves along the lagging strand template. In the codirectional case, by contrast, the front edge of the leading strand DNA polymerase collides with the rear edge of RNA polymerase.While it is unclear a priori which type of collision is more damaging for DNA metabolism, the data on the organization of bacterial genomes point to selection against head-on collisions. Sequencing of the E. coli genome revealed that there is a bias towards codirectional alignment of transcription units with replication (2). Most strikingly, all seven ribosomal operons face the direction of their replichores. For other genes, however, this bias is much less pronounced: ϳ62% of tRNA genes and ϳ55% of protein-coding genes are aligned codirectionally with replication. Similar principles of gene arrangement were observed for other bacteria, such as Bacillus subtilis, Borrelia burgdorferi, Treponema pallidum, Haemophilus influenzae, Helicobacter pylori, Mycoplasma genitalium, and Mycoplasma pneumoniae (36), as well as for bacteriophages T7 and lambda (3).Experimental data on transcription-replication collision are relatively scarce. This problem was addressed in vitro by studying interactions between bacterial RNA polymerases and phage repl...
Collisions between DNA replication and transcription significantly affect genome organization, regulation, and stability. Previous studies have described collisions between replication forks and elongating RNA polymerases. Although replication collisions with the transcription-initiation or -termination complexes are potentially even more important because most genes are not actively transcribed during DNA replication, their existence and mechanisms remained unproven. To address this matter, we have designed a bacterial promoter that binds RNA polymerase and maintains it in the initiating mode by precluding the transition into the elongation mode. By using electrophoretic analysis of replication intermediates, we have found that this steadfast transcriptioninitiation complex inhibits replication fork progression in an orientation-dependent manner during head-on collisions. Transcription terminators also appeared to attenuate DNA replication, but in the opposite, codirectional orientation. Thus, transcription regulatory signals may serve as ''punctuation marks'' for DNA replication in vivo.collisions ͉ promoter ͉ terminator I mpairment of DNA replication is believed to be a major factor in genomic instability (1-13). Because transcription and replication share the same template, occasional collisions between the two machineries are inevitable and can interfere with replication fork progression. Collisions between the elongating RNA polymerase and the replication fork have been well documented in vitro (14-17) and in vivo in both Escherichia coli (18)(19)(20) and Saccharomyces cerevisiae (6,21,22). The consensus from those studies was that head-on collisions with elongating RNA polymerase are much more detrimental for replication fork progression than codirectional collisions. Although it was suggested that replication stalling during the head-on collisions with transcription was caused by topological stress in the DNA separating the two machineries (18,19,22,23), we have recently shown that it is caused by their direct, physical interaction (20). These results, combined with the data on preferred codirectional alignment of transcription units with the direction of replication in prokaryotes (23-27), have led to the suggestion that the main disadvantage of the head-on collisions could be their inhibitory effect on DNA replication.All of the experimental studies cited in the preceding paragraph evaluated the effects of elongating RNA polymerase on the progression of the replication fork. Is there an interplay between the replication machinery and the transcriptioninitiation complex? To the best of our knowledge, there have been few studies on this matter. One intriguing example was the detection of a polar replication fork pause site at the tRNA locus of S. cerevisiae, which depended on the functionality of both the promoter and the RNA polymerase III (pol III) (22). It was believed that the replication fork was attenuated during the encounter with the elongating RNA polymerase (22). A later study, however, suggest...
Schizosaccharomyces pombe Ddb1 is homologous to the mammalian DDB1 protein, which has been implicated in damaged-DNA recognition and global genomic repair. However, a recent study suggested that the S. pombe Ddb1 is involved in cell division and chromosomal segregation. Here, we provide evidence that the S. pombe Ddb1 is functionally linked to the replication checkpoint control gene cds1. We show that the S. pombe strain lacking ddb1 has slow growth due to delayed replication progression. Flow cytometric analysis shows an extensive heterogeneity in DNA content. Furthermore, the ⌬ddb1 strain is hypersensitive to UV irradiation in S phase and is unable to tolerate a prolonged replication block imposed by hydroxyurea. Interestingly, the ⌬ddb1 strain exhibits a high level of the Cds1 kinase activity during passage through S phase. Moreover, mutation of the cds1 gene relieves the defects observed in ⌬ddb1 strain. The results suggest that many of the defects observed in ⌬ddb1 cells are linked to an aberrant activation of Cds1, and that Ddb1 is functionally linked to Cds1.The UV-damaged DNA-binding protein DDB in mammals consists of two subunits, DDB1 and DDB2 (1). DDB has high affinity for UV-induced DNA lesions, cisplatin-modified DNA, and bent DNA (2, 3). A damage sensor function of DDB has been proposed (4). A number of studies indicated a role for DDB in nucleotide excision repair; however, DDB plays a stimulatory but nonessential role in that process (1, 2, 5). The DDB2 gene is mutated in a subset of patients with xeroderma pigmentosum (XP-E), a hereditary disease manifested by sun sensitivity and high susceptibility to skin cancer (4). No mutation in the DDB1 gene has been identified in XP patients. The mammalian DDB1 has been also implicated in other pathways. For example, it was shown to participate in E2F1-activated transcription (6, 7). DDB1 is important in the life cycle of the hepatitis B virus and paramyxovirus (8 -11). It was shown that the HBx protein encoded by hepatitis B virus and the V protein encoded by the paramyxoviruses associate with DDB1 and that these associations are critical for establishment of infection. Interestingly, DDB1, but not DDB2, is conserved in lower eukaryotes, including flies, worms, fission yeast, and slime mold (12).The Schizosaccharomyces pombe and human DDB1 genes share 46% sequence homology, with three domains of 66, 58, and 35% homology that are conserved among other species (12). Markov's model analysis of DDB1 structure predicts a number of -propeller blades comprised of WD-like repeats (13). The position and number of these repeats are remarkably conserved in S. pombe Ddb1 (15 repeats in yeast versus 17 in humans), suggesting that the overall structure and function of the two proteins might be similar. Recently, Zolezzi et al. (14) isolated a cDNA clone of the putative S. pombe orthologue of mammalian DDB1 and characterized the phenotype of an S. pombe strain deficient for ddb1. Their ⌬ddb1 strain, although viable, displays an elongated cell phenotype, abnormal nuc...
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