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Homologous Recombination by the Recbcd and Recf Pathways

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Homologous Recombination by the Recbcd and Recf Pathways The Bactertal Chromosome Edited by N.Patrick Higgins 2005 ASM Press, Washington, D.C. Chapter 21 Homologous Recombination by the RecBCD and RecF Pathways RECOMBINATIONAL REPAIR specific for a particular DNA lesion (see reference 28 OF DNA DAMAGE for a review). These repair systems use the intact com- plementary strand as a template to restore the dam- Homologous, or general, recombination is a cru- aged DNkmolecule to its original state. Occasionally cial biological process that involves the paring and both strands of the double-stranded DNA (dsDNA) transfer of strands between DNA molecules that share can be broken opposite to each other, resulting in a a region of significant sequence homology. After its double-strand break (DSB). DSBs can be produced, discovery in 1946 by Lederberg and Tatum (48), ho- for example, as a direct consequence of ionizing ra- mologous recombination in bacteria was associated diation (Fig. 1A). However, the bulk of the DSBs with the sexual process of conjugation and was viewed in bacteria are generated indirectly as the result of as an evolutionary mechanism both for shuffling the DNA replication through an unrepaired break in just genome and for spreading favorable alleles. However, a single strand of DNA (Fig. 1B). Replication of DNA more recently, a more immediate function of homol- containing a single-strand nick or a gap in the leading ogous recombination has been recognized: namely, it strand results in the dissociation of the DNA poly- is a mechanism for the maintenance of chromosomal merase holoenzyme complex and the generation of integrity that acts to repair DNA lesions, both double- one blunt DSB (Fig. IB), whereas replication of DNA strand DNA breaks and single-strand DNA gaps, gen- with a nick in the lagging strand produces a DSB with erated during the course of DNA replication. The a 3'-terminated single-stranded DNA (ssDNA) tail. intimate connection between the processes of replica- A DSB can also be produced when the DNA tion and recombination was initially appreciated in the replication process is halted by anything that might life cycle of bacteriophage T4 (56) and then later rec- block the progress of the replisome. Upon dissocia- ognized as an important determinant of viability in tion of the replisome, the stalled replication fork can bacteria (39,45).In T4 phage, recombination is linked regress to produce a Holliday junction. Such structure to replication to produce a high yield of phage DNA; contains both a dsDNA end and an intermediate in Escherichia coli, recombination is linked to repli- of reconibination and, hence, attracts recombination cation to permit its completion when interrupted by machinery in a manner similar to that of the simple DNA damage, and also to initiate DNA replication in DSB. In addition, the Holliday junction can be cleaved the absence of origin function. This view of recom- to provide yet another path for DSB formation (Fig. 2). bination as an integral part of efficient chromosome Left unrepaired, DSBs are lethal, and E. coli can re- duplication also reconciled the high level of inviability pair only a few DSBs per chromosome without dying (up to 95%) of recombination-deficient cells (12). (43). Thus, DNA replication is a major source of endogenous DSBs that, in turn, are repaired by ho- mologous recombination. This relationship between GENERATION OF A DSB IN DNA DNA replication and recombinational repair of DSBs is summarized in Fig. 1. A significant fraction of DNA damage affects Recombinational repair, however, requires a only one strand of the DNA duplex. Such lesions can homologous DNA molecule to be used as a template often be repaired by one of the repair systems that are from which to restore, by DNA synthesis, the genetic Maria Spies and Stephen C. Kowalczykowski . Sections of Microbiology and of Molecular and Cellular Biology, Center for Genetics and Development, University of California, Davis, CA 95616-8665. A) directly induced DSBs B) replication-dependent DSBs C) replication-dependent SSGs nick or gap in the nick or gap in the leading no,nsodinglesion in the non-coding lesion in the leading strand template strand template leading strand template leading strand template I I .... DSB i DSB I C DNA strand invasion 1 strand invasion; DNA strand invasion; \ heteroduplex extension heteroduplex extension replication ; 1 mica";/ mica";/ restart \ nuclease 1 replication restart Holliday junction r.01.tion resolution\ Jhet:,"d;p1ex , . extension DSB is repaired; replication restarts Figure 1. Recombinational repair of DNA damage. The DNA strands that are used as templates for the leading strand synthesis are shown in black, and the strands used as templates for the lagging strand synthesis are shown in gray. Arrows indicate the direction of DNA synthesis. (A) Recombinational repair of directly induced DSBs. A DSB, which occurs in newly synthesized DNA, can be repaired by completion of the following steps. First, the DSB is processed to produce 3'-termmated ssDNA. Then, one of the ssDNA tails can invade the homologous dsDNA daughter, displacing one of the resident strands to form a D-loop. This structure can be used as a template for DNA synthesis, ultimately resulting in the formation of a Holliday junction. Upon resolution of the Holliday junction, the replication fork is restored to its original form. When a DSB occurs in a part of the chromosome that is not yet replicated, there is no homologous DNA to serve as a template, and such a DSB can be lethal. (B) Recombinational repair of replication-dependent DSBs. DSBs can be produced by CHAPTER 21 HOMOLOGOUS RECOMBINATION IN E. Cot1 391 >DNA replication block replication fork reversal w regressed replication fork/ Holliday Junction dsDNA end recession J \Holliday Junction cleavage replication fork reassembly J. DNA strand invasion; DNA heteroduplex extension Figure 2. DSBs can be produced as a result of Holliday junction cleavage. Reversal of a stalled DNA replication fork results in the formation of a regressed replication fork, which is a four-way (Holliday) junction that contains a dsDNA end. There are two potential fates for this regressed replication fork: it can be degraded by the recombination machinery to produce a three- way junction that resembles a replication fork, or the Holliday junction can be cleaved to produce a DSB that is repaired by a DSB repair pathway. replication through a single-strand DNA break. The source of the replication-dependent DSBs is a nick or an ssDNA gap in one of the strands of the replicated DNA molecule. Replication through the strand discontinuity results in the formation of one intact and one broken DNA molecule. The end of the broken cliromosome is processed to form a 3'-terminated ssDNA tail, which invades the intact homologous DNA to form a D-loop, which can then be used to restore a normal replication fork. (C) Recombinational repair of'replication-dependent SSGs. SSGs can be produced when synthesis of only one DNA strand is halted by an encounter of a noncoding lesion in that DNA strand. After DNA strand exchange of the ssDNA in the gap with a strand in theintact daughter homologue, the displaced DNA strand can be used as a template for DNA synthesis, resulting in the restoration of the replication fork. Upon completion of replication, one of the DNA molecules will still contain the original DNA damage. If this damage is not repaired by the appropriate repair system, then an SSG will be formed again by the next round of DNA replication. 392 SPES AND KOWALCZYKOWSKI content of the damaged DNA molecule. These ho- The interstrand DNA cross-link is a special class mologous DNA sequences are generated by DNA of chemical damage to DNA, since it blocks DNA replication, and, therefore, the meridiploid character replication completely by preventing DNA strand of bacterial chromosomes provides the necessary separation (Fig. 3) (28).Repair of this special class of templates for recombinational repair. DNA damage is absolutely dependent on homolo- gous recombination (65). Even a single cross-link is lethal for recombination-deficient cells, while wild- GENERATION OF A SINGLE-STRAND type E. coli cells can tolerate up to 70 cross-links GAP IN DNA per chromosome (65). DNA cross-links are repaired through an incisional-recombinational mechanism. An When synthesis of a DNA strand is blocked by a incision is made at both sides of the cross-link on one noncoding lesion (for example, an abasic site or an DNA strand, and displacement of the incised oli- intrastrand cross-link, such as a thymine dimer), gonucleotide results in an SSG that can be repaired continued replication of the flawless strand beyond through an SSG repair pathway. Subsequent incision the lesion produces a single-strand DNA gap (SSG) on the second strand releases the cross-linked strands, (Fig. 1C) (42, 54). SSGs are also known as daughter- producing a second SSG that is repaired by a second strand gaps, since they appear on one of the newly round of recombinational repair. synthesized daughter strands during semiconserva- For all SSGs, if the ssDNA in the gap is cleaved tive DNA replication. before the completion of SSG repair, then the lesion + W+psoralens; bi-functional alkylating agents I I NER SSG NER NER SSG repair I + homologous DSB repair DSB r;l &DNA ' Figure 3. Interstrand cross-links are converted into DSBs and SSGs. Schematic representation for the incision-recombination mechanism of interstrand DNA cross-link repair. The repair of an interstrand cross-link depends on both the nucleotide excision repair (NER) and recombination machineries. The NER enzymes make an incision on either side of the lesion on one DNA strand and also displace the cross-link-containing oligonucleotide to produce an SSG; this SSG can be repaired by an SSG repair pathway.
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