The Disposition of Nascent Strands at Stalled Replication Forks Dictates the Pathway of Replisome Loading during Restart
ABSTRACT Rescue of arrested and collapsed replication forks is essential for maintenance of genomic integrity. One system for origin of replication-independent loading of the DnaB replicative helicase and subsequent replisome reassembly requires the structure-specific recognition factor PriA and the assembly factors PriB and DnaT. Here, we provide biochemical evidence for an alternate system for DnaB loading that requires only PriC. Furthermore, the choice of which system is utilized during restart is dictated by the nature of the structure of the stalled replication fork. PriA-dependent reactions are most robust on fork structures with no gaps in the leading strand, such as is found at the junction of a D loop, while the PriC-dependent system preferentially utilizes fork structures with large gaps in the leading strand. These observations suggest that the type of initial damage on the DNA template and how the inactivated fork is processed ultimately influence the choice of enzymatic restart pathway.
- SourceAvailable from: Aisha H Syeda
- "The importance of this replisome reassembly is underlined by the inviability of cells lacking PriA and a second structure-specific initiator PriC (Sandler 2000). PriA and PriC have complementary DNA substrate specificities, binding preferentially to forked DNA with and without a leading strand present at the branch point, respectively (Heller and Marians 2005b). The inviability of cells lacking both enzymes provides direct evidence for the low probability of replisomes assembled at oriC being able to complete chromosome duplication. "
Article: Recombination and Replication[Show abstract] [Hide abstract]
ABSTRACT: The links between recombination and replication have been appreciated for decades and it is now generally accepted that these two fundamental aspects of DNA metabolism are inseparable: Homologous recombination is essential for completion of DNA replication and vice versa. This review focuses on the roles that recombination enzymes play in underpinning genome duplication, aiding replication fork movement in the face of the many replisome barriers that challenge genome stability. These links have many conserved features across all domains of life, reflecting the conserved nature of the substrate for these reactions, DNA.Cold Spring Harbor perspectives in biology 10/2014; 6(11). DOI:10.1101/cshperspect.a016550
[Show abstract] [Hide abstract]
- "Standard pathways for homologous recombination (RecBC and RecFOR) involve early events that process breaks and gaps leading to RecA-catalyzed strand invasion. This produces Holliday junctions that are moved on recombining duplexes by RuvAB (Heller and Marians 2005; reviewed by West 2003). The later events that stabilize and ultimately resolve these Holliday structures are achieved by two alternative enzymes: (1) the RuvABC resolvasome and (2) the RecG branch-migration enzyme (Benson et al. 1991; Wardrope and Leach 2009). "
ABSTRACT: Tandem genetic duplications arise frequently between the seven directly repeated 5.5kb rrn loci that encode ribosomal RNAs in Salmonella enterica. The closest rrn genes, rrnB and rrnE, flank a 40kb region that includes the purHD operon. Duplications of purHD arise by exchanges between rrn loci and form at high rate (10(-3)/cell/division) that remains high in strains blocked for early steps in recombination (recA, recB and/or recF), but drops 30-fold in mutants blocked for later Holliday junction resolution (ruvC recG). The duplication defect of a ruvC recG mutant was fully corrected by an added mutation in any one of the recA, recB, or recF genes. To explain these results, we propose that early recombination defects activate an alternative single-strand annealing pathway for duplication formation. In wild type cells, rrn duplications form primarily by the action of RecFORA on single strand gaps. Double strand breaks cannot initiate rrn duplications because rrn loci lack Chi sites, which are essential for recombination between two separated rrn sequences. A recA or recF mutation allows unrepaired gaps to accumulate such that different rrn loci can provide single stranded rrn sequences that lack the RecA coating that normally inhibits annealing. A recB mutation activates annealing by allowing double strand ends within rrn to avoid digestion by RecBCD and provide a new source of rrn ends for use in annealing. The equivalent high rates of rrn duplication by recombination and annealing pathways may reflect a limiting economy of gaps and breaks arising in heavily transcribed, palindrome-rich rrn sequences.Genetics 01/2014; 196(1):119-135. DOI:10.1534/genetics.113.158519
[Show abstract] [Hide abstract]
- "Once DnaB has been loaded, the DNA polymerase III replisome and DNA primase are believed to reassemble automatically, via interactions with DnaB. E. coli uses two systems for replication restart, which depend on the nature of the substrates presented (Heller and Marians, 2005). For structures in which a 3 end, equivalent to the newly synthesized leading strand, abuts the fork, a PriA system is used to recruit DnaB and its escort proteins DnaC and DnaT. "
ABSTRACT: The genetics and biochemistry of genetic recombination in E. coli has been studied for over four decades and provides a useful model system to understand recombination in other organisms. Here we provide an overview of the mechanisms of recombination and how such processes contribute to DNA repair. We describe the E. coli functions that are known to contribute to these mechanisms, step by step, and summarize their biochemical properties in relation to the role these proteins play in vivo. We feature areas of investigation that are newly emerging, as well as work that provides a historical perspective to the field. Finally, we highlight some of the questions that remain unanswered.Critical Reviews in Biochemistry and Molecular Biology 12/2008; 43(6):347-70. DOI:10.1080/10409230802485358