How RecBCD Enzyme and Chi Promote DNA Break Repair and Recombination: a Molecular Biologist's View

Smith, Gerald R.

doi:10.1128/mmbr.05026-11

Cited by 155 publications

(195 citation statements)

References 114 publications

(86 reference statements)

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“…Even though formation of pince-nez chromosomes had been already proposed (Russo et al 1992;Smith 2012), and even reported in plasmids (Petes and Williamson 1994), possible mechanisms of their resolution are currently unknown. Thus, until such resolution mechanisms are found, tying up the replicating chromosome mass in pince-nez structures may be considered a lethal event and a general explanation for the toxicity of maximized CRC in recombinational repair-proficient cells.…”

Section: Recombinational Misrepairmentioning

confidence: 99%

Static and Dynamic Factors Limit Chromosomal Replication Complexity inEscherichia coli, Avoiding Dangers of Runaway Overreplication

Khan¹,

Mahaseth²,

Kouzminova³

et al. 2016

Genetics

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We define chromosomal replication complexity (CRC) as the ratio of the copy number of the most replicated regions to that of unreplicated regions on the same chromosome. Although a typical CRC of eukaryotic or bacterial chromosomes is 2, rapidly growing Escherichia coli cells induce an extra round of replication in their chromosomes (CRC = 4). There are also E. coli mutants with stable CRC6. We have investigated the limits and consequences of elevated CRC in E. coli and found three limits: the "natural" CRC limit of 8 (cells divide more slowly); the "functional" CRC limit of 22 (cells divide extremely slowly); and the "tolerance" CRC limit of 64 (cells stop dividing). While the natural limit is likely maintained by the eclipse system spacing replication initiations, the functional limit might reflect the capacity of the chromosome segregation system, rather than dedicated mechanisms, and the tolerance limit may result from titration of limiting replication factors. Whereas recombinational repair is beneficial for cells at the natural and functional CRC limits, we show that it becomes detrimental at the tolerance CRC limit, suggesting recombinational misrepair during the runaway overreplication and giving a rationale for avoidance of the latter. KEYWORDS overinitiation; hydroxyurea; seqA; rep; recA E UKARYOTIC and prokaryotic chromosomes differ in many important aspects (Kuzminov 2014), and one key difference lies in the spatio-temporal organization of chromosomal replication. In contrast to eukaryotes, which perform multibubble replication (Masai et al. 2010), most bacteria replicate their singular chromosome in the unibubble format by initiating bidirectional replication from a designated replication origin (oriC) (Sernova and Gelfand 2008;Leonard and Méchali 2013) and finishing replication within a broad termination zone (ter) (Mirkin and Mirkin 2007;Duggin et al. 2008). Eukaryotes always perform a single replication round in their chromosomes (Masai et al. 2010;Diffley 2011), keeping the ratio of maximally replicated to unreplicated DNA in the same chromosome (the "replication complexity index") strictly at 2 ( Figure 1A). Due to the defined origin and terminus of prokaryotic chromosomes, chromosomal replication complexity in bacteria can be simply expressed as the ori/ter ratio ( Figure 1B). Even though unique cell cycles in some bacteria, such as Caulobacter, also maintain a strict CRC = 2 (Collier 2012), bacterial cells are generally recognized for their ability to support multiple concurrent replication rounds within the same chromosome (Morigen et al. 2009).In reality, under typical growth conditions the ori/ter ratio in exponentially growing bacterial cells is still 2 (Bird et al. 1972;Bipatnath et al. 1998;Wang et al. 2007;Murray and Errington 2008;Rotman et al. 2009;Stokke et al. 2011), showing that bacterial cells, like eukaryotes, also prefer to deal with a single replication round in their chromosomes. However, due to the peculiarity of the prokaryotic chromosome cycle (Kuzminov 2013), whe...

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Section: Recombinational Misrepairmentioning

confidence: 99%

Static and Dynamic Factors Limit Chromosomal Replication Complexity inEscherichia coli, Avoiding Dangers of Runaway Overreplication

Khan¹,

Mahaseth²,

Kouzminova³

et al. 2016

Genetics

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“…The newly generated 39 ss end, with Chi near its end, is coated by RecA strand-exchange protein and invades an intact homologous duplex to generate a D-loop. The D-loop is converted into a Holliday junction, which may be resolved into reciprocal recombinants; alternatively, the D-loop may prime DNA replication and generate nonreciprocal recombinants (Smith 1991(Smith , 2012. Later reports showed that RecBCD actively loads RecA onto the newly generated 39 end in a Chi-dependent manner (Anderson and Kowalczykowski 1997).…”

mentioning

confidence: 99%

“…The sites controlling homologous recombination that are best understood at the molecular level are the Chi hotspots of Escherichia coli (Smith 2012). Chi sites control RecBCD enzyme, a complex enzyme with both nuclease and helicase activities essential for the major pathway of DNA double-strand break repair and genetic recombination.…”

mentioning

confidence: 99%

RecBCD Enzyme “Chi Recognition” Mutants Recognize Chi Recombination Hotspots in the Right DNA Context

2016

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RecBCD enzyme is a complex, three-subunit protein machine essential for the major pathway of DNA double-strand break repair and homologous recombination in Escherichia coli. Upon encountering a Chi recombination-hotspot during DNA unwinding, RecBCD nicks DNA to produce a single-stranded DNA end onto which it loads RecA protein. Conformational changes that regulate RecBCD's helicase and nuclease activities are induced upon its interaction with Chi, defined historically as 59 GCTGGTGG 39. Chi is thought to be recognized as single-stranded DNA passing through a tunnel in RecC. To define the Chi recognition-domain in RecC and thus the mechanism of the RecBCD-Chi interaction, we altered by random mutagenesis eight RecC amino acids lining the tunnel. We screened for loss of Chi activity with Chi at one site in bacteriophage l. The 25 recC mutants analyzed thoroughly had undetectable or strongly reduced Chi-hotspot activity with previously reported Chi sites. Remarkably, most of these mutants had readily detectable, and some nearly wild-type, activity with Chi at newly generated Chi sites. Like wild-type RecBCD, these mutants had Chi activity that responded dramatically (up to fivefold, equivalent to Chi's hotspot activity) to nucleotide changes flanking 59 GCTGGTGG 39. Thus, these and previously published RecC mutants thought to be Chi-recognition mutants are actually Chi context-dependence mutants. Our results fundamentally alter the view that Chi is a simple 8-bp sequence recognized by the RecC tunnel. We propose that Chi hotspots have dual nucleotide sequence interactions, with both the RecC tunnel and the RecB nuclease domain.KEYWORDS chromosomal sites; recombination hotspots; Chi; RecBCD enzyme; DNA context-dependence H OMOLOGOUS recombination, like other aspects of chromosome metabolism, is controlled by special DNA sites. The sites controlling homologous recombination that are best understood at the molecular level are the Chi hotspots of Escherichia coli (Smith 2012). Chi sites control RecBCD enzyme, a complex enzyme with both nuclease and helicase activities essential for the major pathway of DNA double-strand break repair and genetic recombination. These sites, called Chi for crossover hotspot instigator (Lam et al. 1974), locally stimulate recombination by altering the multiple activities of RecBCD. Here, we address the question of how RecBCD recognizes Chi, the first step in its stimulation of recombination. Our results force a reanalysis of both the Chi nucleotide sequence and how RecBCD recognizes Chi.Chi was discovered as a set of mutations that increase the plaque size of phage l red gam mutants, which rely on the E. coli RecBCD pathway for growth (Lam et al. 1974;McMilin et al. 1974;Henderson and Weil 1975). In the absence of its own recombination functions (Red) and of the RecBCD inhibitor (Gam), l replication forms only monomeric circles. Phage DNA packaging, however, requires dimeric or higher order concatemers, which can be formed by RecBCDpromoted recombination between monomers. Wild-typ...

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“…RecBCD processes double-stranded DNA breaks during homologous recombination and replication fork rescue in Escherichia coli and also degrades linear chromosome fragments to prevent aberrant DNA replication or recombination (10,11,14). Interestingly, RecB and RecC are the only two recombination proteins necessary for cell viability when head-on replication-transcription collisions are exacerbated by inversion of the rRNA operon (15).…”

mentioning

confidence: 99%

“…RecBCD is a large (330-kDa) heterotrimeric complex that has served as an important model system for understanding the properties of nucleic acid motor proteins (10)(11)(12)(13). RecBCD processes double-stranded DNA breaks during homologous recombination and replication fork rescue in Escherichia coli and also degrades linear chromosome fragments to prevent aberrant DNA replication or recombination (10,11,14).…”

mentioning

confidence: 99%

Sequential eviction of crowded nucleoprotein complexes by the exonuclease RecBCD molecular motor

Terakawa

Redding

Silverstein

et al. 2017

Proc. Natl. Acad. Sci. U.S.A.

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In physiological settings, all nucleic acids motor proteins must travel along substrates that are crowded with other proteins. However, the physical basis for how motor proteins behave in these highly crowded environments remains unknown. Here, we use real-time single-molecule imaging to determine how the ATPdependent translocase RecBCD travels along DNA occupied by tandem arrays of high-affinity DNA binding proteins. We show that RecBCD forces each protein into its nearest adjacent neighbor, causing rapid disruption of the protein-nucleic acid interaction. This mechanism is not the same way that RecBCD disrupts isolated nucleoprotein complexes on otherwise naked DNA. Instead, molecular crowding itself completely alters the mechanism by which RecBCD removes tightly bound protein obstacles from DNA.RecBCD | single molecule | DNA curtain | molecular motor | molecular crowding L ong stretches of naked DNA do not exist in living cells. Instead, chromosomes are bound by all of the proteins that are necessary for genome compaction, organization, regulation, and maintenance. DNA polymerases, RNA polymerases (RNAPs), helicases, translocases, and chromatin remodeling complexes must all travel along the highly crowded nucleic acids that exist within these physiological settings. There is a growing appreciation that ATP-dependent motor proteins are required to either remove or remodel nucleoprotein complexes that may otherwise block normal processes related to nucleic acid metabolism, including DNA replication, transcription, and DNA repair (1-9). Despite this importance, there remains almost no detailed mechanistic information describing how molecular motor proteins of any type behave on highly crowded nucleic acids.RecBCD is a large (330-kDa) heterotrimeric complex that has served as an important model system for understanding the properties of nucleic acid motor proteins (10-13). RecBCD processes double-stranded DNA breaks during homologous recombination and replication fork rescue in Escherichia coli and also degrades linear chromosome fragments to prevent aberrant DNA replication or recombination (10,11,14). Interestingly, RecB and RecC are the only two recombination proteins necessary for cell viability when head-on replication-transcription collisions are exacerbated by inversion of the rRNA operon (15). In addition to its roles in protecting genome integrity, RecBCD is also a self-defense enzyme that degrades foreign invaders, such as bacteriophage, and the resulting DNA fragments are incorporated into the CRISPR locus, providing immunity against additional infection (16). RecBCD possesses two ATP-dependent Superfamily 1 (SF1) molecular motor proteins, the 3′→5′ SF1A helicase RecB (134 kDa) and the 5′→3′ SF1B helicase RecD (67 kDa) (13). The RecC subunit (129 kDa) holds the complex together and coordinates the response to 8-nt cis-acting cross-over hotspot instigator (Chi) sequences (5′-dGCTGGTGG-3′). RecD is the lead motor before Chi, RecB is the lead motor after Chi, and Chi recognition is accompanied by a reduce...

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How RecBCD Enzyme and Chi Promote DNA Break Repair and Recombination: a Molecular Biologist's View

Cited by 155 publications

References 114 publications

Static and Dynamic Factors Limit Chromosomal Replication Complexity inEscherichia coli, Avoiding Dangers of Runaway Overreplication

Static and Dynamic Factors Limit Chromosomal Replication Complexity inEscherichia coli, Avoiding Dangers of Runaway Overreplication

RecBCD Enzyme “Chi Recognition” Mutants Recognize Chi Recombination Hotspots in the Right DNA Context

Sequential eviction of crowded nucleoprotein complexes by the exonuclease RecBCD molecular motor

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