Genome maintenance is a constant concern for cells, and a coordinated response to DNA damage is required to maintain cellular viability and prevent disease. The ataxia-telangiectasia mutated (ATM) and ATM and RAD3-related (ATR) protein kinases act as master regulators of the DNA-damage response by signalling to control cell-cycle transitions, DNA replication, DNA repair and apoptosis. Recent studies have provided new insights into the mechanisms that control ATR activation, have helped to explain the overlapping but non-redundant activities of ATR and ATM in DNA-damage signalling, and have clarified the crucial functions of ATR in maintaining genome integrity.

/pdf/atr-an-essential-regulator-of-genome-integrity-3i2zlw33w3.pdf

ATR: an essential regulator of genome integrity

https://www.cell.com/molecular-cell/pdf/S1097-2765(12)00305-X.pdf

R Loops: From Transcription Byproducts to Threats to Genome Stability

Aberrant DNA replication is a major source of the mutations and chromosome rearrangements that are associated with pathological disorders. When replication is compromised, DNA becomes more prone to breakage. Secondary structures, highly transcribed DNA sequences and damaged DNA stall replication forks, which then require checkpoint factors and specialized enzymatic activities for their stabilization and subsequent advance. These mechanisms ensure that the local DNA damage response, which enables replication fork progression and DNA repair in S phase, is coupled with cell cycle transitions. The mechanisms that operate in eukaryotic cells to promote replication fork integrity and coordinate replication with other aspects of chromosome maintenance are becoming clear.

Maintaining genome stability at the replication fork.

Genomic instability in the form of mutations and chromosome rearrangements is usually associated with pathological disorders, and yet it is also crucial for evolution. Two types of elements have a key role in instability leading to rearrangements: those that act in trans to prevent instability--among them are replication, repair and S-phase checkpoint factors--and those that act in cis--chromosomal hotspots of instability such as fragile sites and highly transcribed DNA sequences. Taking these elements as a guide, we review the causes and consequences of instability with the aim of providing a mechanistic perspective on the origin of genomic instability.

Genome instability: a mechanistic view of its causes and consequences

DNA is subject to many endogenous and exogenous insults that impair DNA replication and proper chromosome segregation. DNA double-strand breaks (DSBs) are one of the most toxic of these lesions and must be repaired to preserve chromosomal integrity. Eukaryotes are equipped with several different, but related, repair mechanisms involving homologous recombination, including single-strand annealing, gene conversion, and break-induced replication. In this review, we highlight the chief sources of DSBs and crucial requirements for each of these repair processes, as well as the methods to identify and study intermediate steps in DSB repair by homologous recombination.

/pdf/sources-of-dna-double-strand-breaks-and-models-of-2ej8n7gzfl.pdf

Sources of DNA Double-Strand Breaks and Models of Recombinational DNA Repair

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.

https://mmbr.asm.org/content/mmbr/71/1/13.full.pdf

Replication Fork Stalling at Natural Impediments

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.

/pdf/mechanisms-of-transcription-replication-collisions-in-18y10b8ni0.pdf

Mechanisms of transcription-replication collisions in bacteria.

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.

/pdf/transcription-regulatory-elements-are-punctuation-marks-for-29sqglq0xx.pdf

Transcription regulatory elements are punctuation marks for DNA replication

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

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.

Ekaterina V. Mirkin

Papers

Replication Fork Stalling at Natural Impediments

Mechanisms of transcription-replication collisions in bacteria.

Transcription regulatory elements are punctuation marks for DNA replication

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

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