So similar yet so different: The two ends of a double strand break.

Homologous recombination (HR) is essential for ensuring proper segregation of chromosomes in the first round of meiotic division. HR is also crucial for preserving genomic integrity of somatic cells due to its ability to rescue collapsed replication forks and eliminate deleterious DNA lesions, such as double-strand breaks (DSBs), interstrand crosslinks, and single-strand DNA gaps. Here, we review the early steps of HR (homology search and strand exchange), focusing on the roles of the two ends of a DSB. A detailed overview of the basic HR machinery and its mechanism for template selection and capture of duplex DNA via strand exchange is provided. Roles of proteins involved in these steps are discussed in both mitotic and meiotic HR. Central to this review is the hypothesis, which suggests that in meiosis, HR begins with a symmetrical DSB, but the symmetry is quickly lost with the two ends assuming different roles; it argues that this disparity of the two ends is essential for regulation of HR in meiosis and successful production of haploid gametes. We also propose a possible evolutionary reason for the asymmetry of the ends in HR.

To switch or not to switch: at the origin of repeat expansion disease.

Expansions of DNA repeats cause hereditary disorders in humans. Gerhardt et al. (2014) argue that a developmental switch in the direction of DNA replication through the (CGG)n repeat predisposes it to expansions during intergenerational transmissions leading to fragile X syndrome.

Protein-mediated chromosome pairing of repetitive arrays.

Chromosomally integrated arrays of lacO and tetO operator sites visualized by LacI and TetR repressor proteins fused with GFP (green fluorescent protein) (or other fluorescent proteins) are widely used to monitor the behavior of chromosomal loci in various systems. However, insertion of such arrays and expression of the corresponding proteins is known to perturb genomic architecture. In several cases, juxtaposition of such arrays located on different chromosomes has been inferred to reflect pairing of the corresponding loci. Here, we report that a version of TetR-GFP mutated to disrupt GFP dimerization (TetR-A206KGFP or "TetR-kGFP") abolishes pairing of tetO arrays in vivo and brings spatial proximity of chromosomal loci marked with those arrays back to the wild-type level. These data argue that pairing of arrays is caused by GFP dimerization and thus presents an example of protein-assisted interaction in chromosomes. Arrays marked with another protein, TetR-tdTomato, which has a propensity to form intramolecular dimers instead of intermolecular dimers, also display reduced level of pairing, supporting this idea. TetR-kGFP provides an improved system for studying chromosomal loci with a low pairing background.

Dynamic trans interactions in yeast chromosomes.

Three-dimensional organization of the genome is important for regulation of gene expression and maintenance of genomic stability. It also defines, and is defined by, contacts between different chromosomal loci. Interactions between loci positioned on different chromosomes, i.e. "trans" interactions are one type of such contacts. Here, we describe a case of inducible trans interaction in chromosomes of the budding yeast S. cerevisiae. Special DNA sequences, inserted in two ectopic chromosomal loci positioned in trans, pair with one another in an inducible manner. The spatial proximity diagnostic of pairing is observable by both chromosome capture analysis (3C) and epifluorescence microscopy in whole cells. Protein synthesis de novo appears to be required for this process. The three-dimensional organization of the yeast nucleus imposes a constraint on such pairing, presumably by dictating the probability with which the two sequences collide with one another.

Replication fork stalling at natural impediments.

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.

Transcription regulatory elements are punctuation marks for DNA replication.

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 transcription-initiation 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.

Mechanisms of transcription-replication collisions in bacteria.

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.

Schizosaccharomyces pombe Ddb1 is functionally linked to the replication checkpoint pathway.

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 Deltaddb1 strain is hypersensitive to UV irradiation in S phase and is unable to tolerate a prolonged replication block imposed by hydroxyurea. Interestingly, the Deltaddb1 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 Deltaddb1 strain. The results suggest that many of the defects observed in Deltaddb1 cells are linked to an aberrant activation of Cds1, and that Ddb1 is functionally linked to Cds1.