Peter McGlynn

PARP is activated at stalled forks to mediate Mre11-dependent replication restart and recombination.

If replication forks are perturbed, a multifaceted response including several DNA repair and cell cycle checkpoint pathways is activated to ensure faithful DNA replication. Here, we show that poly(ADP‐ribose) polymerase 1 (PARP1) binds to and is activated by stalled replication forks that contain small gaps. PARP1 collaborates with Mre11 to promote replication fork restart after release from replication blocks, most likely by recruiting Mre11 to the replication fork to promote resection of DNA. Both PARP1 and PARP2 are required for hydroxyurea‐induced homologous recombination to promote cell survival after replication blocks. Together, our data suggest that PARP1 and PARP2 detect disrupted replication forks and attract Mre11 for end processing that is required for subsequent recombination repair and restart of replication forks.

Modulation of RNA Polymerase by (p)ppGpp Reveals a RecG-Dependent Mechanism for Replication Fork Progression

We have discovered a correlation between the ability of Escherichia coli cells to survive damage to DNA and their ability to modulate RNA polymerase via the stringent response regulators, (p)ppGpp. Elevation of (p)ppGpp, or certain mutations in the beta subunit of RNA polymerase, dramatically improve survival of UV-irradiated strains lacking the RuvABC Holliday junction resolvase. Increased survival depends on excision and recombination proteins and relies on the ability of RecG helicase to form Holliday junctions from replication forks stalled at lesions in the DNA and of PriA to initiate replication restart. The role of RecG provides novel insights into the interplay between transcription, replication, and recombination, and suggests a general model in which recombination underpins genome duplication in the face of frequent obstacles to replication fork progression.

Rescue of stalled replication forks by RecG: Simultaneous translocation on the leading and lagging strand templates supports an active DNA unwinding model of fork reversal and Holliday junction formation

Modification of damaged replication forks is emerging as a crucial factor for efficient chromosomal duplication and the avoidance of genetic instability. The RecG helicase of Escherichia coli, which is involved in recombination and DNA repair, has been postulated to act on stalled replication forks to promote replication restart via the formation of a four-stranded (Holliday) junction. Here we show that RecG can actively unwind the leading and lagging strand arms of model replication fork structures in vitro. Unwinding is achieved in each case by simultaneous interaction with and translocation along both the leading and lagging strand templates at a fork. Disruption of either of these interactions dramatically inhibits unwinding of the opposing duplex arm. Thus, RecG translocates simultaneously along two DNA strands, one with 5′-3′ and the other with 3′-5′ polarity. The unwinding of both nascent strands at a damaged fork, and their subsequent annealing to form a Holliday junction, may explain the ability of RecG to promote replication restart. Moreover, the preferential binding of partial forks lacking a leading strand suggests that RecG may have the ability to target stalled replication intermediates in vivo in which lagging strand synthesis has continued beyond the leading strand.

Replication fork reversal and the maintenance of genome stability

The progress of replication forks is often threatened in vivo, both by DNA damage and by proteins bound to the template. Blocked forks must somehow be restarted, and the original blockage cleared, in order to complete genome duplication, implying that blocked fork processing may be critical for genome stability. One possible pathway that might allow processing and restart of blocked forks, replication fork reversal, involves the unwinding of blocked forks to form four-stranded structures resembling Holliday junctions. This concept has gained increasing popularity recently based on the ability of such processing to explain many genetic observations, the detection of unwound fork structures in vivo and the identification of enzymes that have the capacity to catalyse fork regression in vitro. Here, we discuss the contexts in which fork regression might occur, the factors that may promote such a reaction and the possible roles of replication fork unwinding in normal DNA metabolism.

Formation of Holliday junctions by regression of nascent DNA in intermediates containing stalled replication forks: RecG stimulates regression even when the DNA is negatively supercoiled

Replication forks formed at bacterial origins often encounter template roadblocks in the form of DNA adducts and frozen protein–DNA complexes, leading to replication-fork stalling and inactivation. Subsequent correction of the corrupting template lesion and origin-independent assembly of a new replisome therefore are required for survival of the bacterium. A number of models for replication-fork restart under these conditions posit that nascent strand regression at the stalled fork generates a Holliday junction that is a substrate for subsequent processing by recombination and repair enzymes. We show here that early replication intermediates containing replication forks stalled in vitro by the accumulation of excess positive supercoils could be cleaved by the Holliday junction resolvases RusA and RuvC. Cleavage by RusA was inhibited by the presence of RuvA and was stimulated by RecG, confirming the presence of Holliday junctions in the replication intermediate and supporting the previous proposal that RecG could catalyze nascent strand regression at stalled replication forks. Furthermore, RecG promoted Holliday junction formation when replication intermediates in which the replisome had been inactivated were negatively supercoiled, suggesting that under intracellular conditions, the action of RecG, or helicases with similar activities, is necessary for the catalysis of nascent strand regression.

Genome stability and the processing of damaged replication forks by RecG

Chromosomal duplication faces many blocks to replication fork progression that could destabilize the genome and prove fatal if not overcome. Overcoming such blocks requires interplay between DNA replication, recombination and repair. The RecG protein of Escherichia coli promotes rescue of damaged forks by catalysing their unwinding and conversion to Holliday junctions. Subsequent processing of this structure allows repair or bypass of the fork block, enabling replication to resume without recourse to potentially mutagenic translesion synthesis or recombination. Such direct rescue of stalled forks might help safeguard genome integrity in all organisms.

Direct Rescue of Stalled DNA Replication Forks via the Combined Action of PriA and RecG Helicase Activities

The PriA protein of Escherichia coli plays a key role in the rescue of replication forks stalled on the template DNA. One attractive model of rescue relies on homologous recombination to establish a new fork via PriA-mediated loading of the DnaB replicative helicase at D loop intermediates. We provide genetic and biochemical evidence that PriA helicase activity can also rescue a stalled fork by an alternative mechanism that requires manipulation of the fork before loading of DnaB on the lagging strand template. This direct rescue depends on RecG, which unwinds forks and Holliday junctions and interconverts these structures. The combined action of PriA and RecG helicase activities may thus avoid the potential dangers of rescue pathways involving fork breakage and recombination.

Rep Provides a Second Motor at the Replisome to Promote Duplication of Protein-Bound DNA

Summary Nucleoprotein complexes present challenges to genome stability by acting as potent blocks to replication. One attractive model of how such conflicts are resolved is direct targeting of blocked forks by helicases with the ability to displace the blocking protein-DNA complex. We show that Rep and UvrD each promote movement of E. coli replisomes blocked by nucleoprotein complexes in vitro, that such an activity is required to clear protein blocks (primarily transcription complexes) in vivo, and that a polarity of translocation opposite that of the replicative helicase is critical for this activity. However, these two helicases are not equivalent. Rep but not UvrD interacts physically and functionally with the replicative helicase. In contrast, UvrD likely provides a general means of protein-DNA complex turnover during replication, repair, and recombination. Rep and UvrD therefore provide two contrasting solutions as to how organisms may promote replication of protein-bound DNA.

The purple bacterial photosynthetic unit

Now is a very exciting time for researchers in the area of the primary reactions of purple bacterial photosynthesis. Detailed structural information is now available for not only the reaction center (Lancaster et al. 1995, in: Blankenship RE et al. (eds) Anoxygenic Photosynthetic Bacteria, pp 503–526), but also LH2 from Rhodopseudomonas acidophila (McDermott et al. 1995, Nature 374: 517–521) and LH1 from Rhodospirillum rubrum (Karrasch et al. 1995. EMBO J 14: 631–638). These structures can now be integrated to produce models of the complete photosynthetic unit (PSU) (Papiz et al., 1996, Trends Plant Sci, in press), which opens the door to a much more detailed understanding of the energy transfer events occurring within the PSU.

The Rhodobacter sphaeroides PufX protein is not required for photosynthetic competence in the absence of a light harvesting system

The effects of deletion of the gene encoding the PufX protein from Rhodobacter sphaeroides have been examined using bacterial strains with simplified photosystems. We find that the PufX protein is required for photosynthetic growth in strains which have the LH1 antenna complex, but is not required in a reaction centre‐only strain, suggesting that the PufX protein does not directly facilitate cyclic electron transfer between the reaction centre and the cytochrome bc 1 complex. The influence of PufX and carotenoid type on the size of the reaction center/LH1 core complex has also been examined in these strains.