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

Abstract

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...

CRISPR–Cas adaptation in Escherichia coli requires RecBCD helicase but not nuclease activity, is independent of homologous recombination, and is antagonized by 5′ ssDNA exonucleases

“…The adaptation phenotypes associated with recBCD might indicate that DNA pre-processing and capture for naïve adaptation requires DNA translocation unwinding associated with RecB. RecBCD is a powerful translocase that can clear DNA of RNA polymerase, nucleosome and other DNA bound proteins ( 41 , 42 ). We propose that RecBCD helicase-translocase activities are required for adaptation to disrupt or displace nucleoprotein complexes present at DNA capture sites to provide access to DNA for Cas1–Cas2 and generate substrates that can be acted on by Cas1–Cas2 for DNA capture (Figure 6 ).…”

Direct observation of end resection by RecBCD during double-stranded DNA break repair in vivo

“…Although helicases translocating along cellular DNA will necessarily encounter numerous roadblocks such as RNA polymerases or proteins organizing the genome, RecBCD seems not to be hindered by these. Indeed, RecBCD was shown to be able to knock off multiple substrates from the DNA in vitro ( 41 ). Speeds of eukaryotic and prokaryotic end resection were estimated before from indirect measurements such as southern blots, DAPI signal decay or quantitative modeling to fit degradation profiles obtained with high-throughput sequencing.…”

Transcription reinitiation by recycling RNA polymerase that diffuses on DNA after releasing terminated RNA

“…36 This mutant preparation has been shown to bind its cognate sequence very tightly (K d ≈ fM) and has been used as a protein obstacle on DNA in several previous experiments. 18,37,38 Cy5 labeling of σ 70 factor. E. coli RpoD was labeled with Cy5 at the 366th residue Cys substituting Ser, while Cys residues at the 132nd, 291st, and 295th positions had been substituted for Ser.…”