A new trick for an old dog: TraY binding to a homopurine-homopyrimidine run attenuates DNA replication

Krasilnikova, Maria M.; Смирнова, Е В; Krasilnikov, Andrei S; Mirkin, Sergei M.

doi:10.1006/jmbi.2001.5059

Cited by 9 publications

(8 citation statements)

References 59 publications

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“…2C. It is quite different from the repeat-caused replication stalling described by us previously (21,22,43), where distinct stalling sites were observed. Bacterial plasmids, including ours, contain more than one promoter.…”

Section: Resultsmentioning

confidence: 60%

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Mechanisms of Transcription-Replication Collisions in Bacteria

Mirkin

2005

Molecular and Cellular Biology

159

153

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While collisions between replication and transcription in bacteria are deemed inevitable, the fine details of the interplay between the two machineries are poorly understood. In this study, we evaluate the effects of transcription on the replication fork progression in vivo, by using electrophoresis analysis of replication intermediates. Studying Escherichia coli plasmids, which carry constitutive or inducible promoters in different orientations relative to the replication origin, we show that the mutual orientation of the two processes determines their mode of interaction. Replication elongation appears not to be affected by transcription proceeding in the codirectional orientation. Head-on transcription, by contrast, leads to severe inhibition of the replication fork progression. Furthermore, we evaluate the mechanism of this inhibition by limiting the area of direct contact between the two machineries. We observe that replication pausing zones coincide exactly with transcribed DNA segments. We conclude, therefore, that the replication fork is most likely attenuated upon direct physical interaction with the head-on transcription machinery.In bacteria, DNA replication and transcription continue throughout the life cycle. The speed of the replication fork progression in Escherichia coli is ϳ1,000 bp per s (16), while the elongation rate for RNA polymerase is just 50 nucleotides (nt) per s (15); i.e., replication is approximately 20-fold faster than transcription. Since the two processes proceed simultaneously, frequent collisions between replication and transcription seem unavoidable (3, 38). Given that both processes are polar, the collisions can occur either head-on or codirectionally (Fig. 1). In the case of head-on collisions, the front edge of RNA polymerase meets the hexameric DNA helicase DnaB that moves along the lagging strand template. In the codirectional case, by contrast, the front edge of the leading strand DNA polymerase collides with the rear edge of RNA polymerase.While it is unclear a priori which type of collision is more damaging for DNA metabolism, the data on the organization of bacterial genomes point to selection against head-on collisions. Sequencing of the E. coli genome revealed that there is a bias towards codirectional alignment of transcription units with replication (2). Most strikingly, all seven ribosomal operons face the direction of their replichores. For other genes, however, this bias is much less pronounced: ϳ62% of tRNA genes and ϳ55% of protein-coding genes are aligned codirectionally with replication. Similar principles of gene arrangement were observed for other bacteria, such as Bacillus subtilis, Borrelia burgdorferi, Treponema pallidum, Haemophilus influenzae, Helicobacter pylori, Mycoplasma genitalium, and Mycoplasma pneumoniae (36), as well as for bacteriophages T7 and lambda (3).Experimental data on transcription-replication collision are relatively scarce. This problem was addressed in vitro by studying interactions between bacterial RNA polymerases and phage repl...

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Section: Resultsmentioning

confidence: 60%

“…2B). We have previously applied this approach for detecting replication pauses caused by various DNA repeats (21,22,43) and have found it to be extremely reliable and sensitive.…”

Section: Resultsmentioning

confidence: 99%

Mechanisms of Transcription-Replication Collisions in Bacteria

Mirkin

2005

Molecular and Cellular Biology

159

153

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“…Our data exclude aberrant transcription of the repeat. Replication arrest could also be caused by a concerted binding of multiple protein molecules to a repetitive run as described by us for (GA) n ⅐ (TC) n repeats (19). In the latter case, however, replication inhibition was equally pronounced in both orientations of the repeat relative to the ori, as one would expect for protein binding.…”

Section: Discussionmentioning

confidence: 65%

Replication Stalling at Friedreich's Ataxia (GAA)_n Repeats In Vivo

Krasilnikova

Mirkin

2004

Molecular and Cellular Biology

194

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Friedreich's ataxia (GAA) n repeats of various lengths were cloned into a Saccharymyces cerevisiae plasmid, and their effects on DNA replication were analyzed using two-dimensional electrophoresis of replication intermediates. We found that premutation-and disease-size repeats stalled the replication fork progression in vivo, while normal-size repeats did not affect replication. Remarkably, the observed threshold repeat length for replication stalling in yeast (ϳ40 repeats) closely matched the threshold length for repeat expansion in humans. Further, replication stalling was strikingly orientation dependent, being pronounced only when the repeat's homopurine strand served as the lagging strand template. Finally, it appeared that length polymorphism of the (GAA) n ⅐ (TTC) n repeat in both expansions and contractions drastically increases in the repeat's orientation that is responsible for the replication stalling. These data represent the first direct proof of the effects of (GAA) n repeats on DNA replication in vivo. We believe that repeat-caused replication attenuation in vivo is due to triplex formation. The apparent link between the replication stalling and length polymorphism of the repeat points to a new model for the repeat expansion.

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“…Our earlier work revealed an enhancement of negative supercoiling by the TraD-related T4CP TrwB (52). The F TraY protein increases bending of F oriT (47) and is believed to wrap DNA into nucleosome-like structures when bound to simple repeats of d(GA) n /d(TC) n on unrelated molecules (37). Extreme planar bending is a hallmark of the IHF interaction with DNA (61).…”

Section: Discussionmentioning

confidence: 99%

Plasmid R1 Conjugative DNA Processing Is Regulated at the Coupling Protein Interface

et al. 2009

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Selective substrate uptake controls initiation of macromolecular secretion by type IV secretion systems in gram-negative bacteria. Type IV coupling proteins (T4CPs) are essential, but the molecular mechanisms governing substrate entry to the translocation pathway remain obscure. We report a biochemical approach to reconstitute a regulatory interface between the plasmid R1 T4CP and the nucleoprotein relaxosome dedicated to the initiation stage of plasmid DNA processing and substrate presentation. The predicted cytosolic domain of T4CP TraD was purified in a predominantly monomeric form, and potential regulatory effects of this protein on catalytic activities exhibited by the relaxosome during transfer initiation were analyzed in vitro. Type IV secretion systems (T4SS) in gram-negative bacteria mediate translocation of macromolecules out of the bacterial cell (14). The transmission of effector proteins and DNA into plant cells or other bacteria via cell-cell contact is one example of their function, and conjugation systems as well as the transferred DNA (T-DNA) delivery system of the phytopathogen Agrobacterium tumefaciens are prototypical of the T4SS family. Macromolecular translocation is achieved by a membranespanning protein machinery comprised of 12 gene products, VirB1 to VirB11 and an associated factor known as the coupling protein (VirD4) (66). The T4SS-associated coupling protein (T4CP) performs a crucial function in recognition of appropriate secretion substrates and governing entry of those molecules to the translocation pathway (7,8,10,30,41). In conjugation systems substrate recognition is applied to the relaxosome, a nucleoprotein complex of DNA transfer initiator proteins assembled specifically at the plasmid origin of transfer (oriT). In current models, initiation of the reactions that provide the single strand of plasmid (T-strand) DNA for secretion to recipient bacteria is expected to resemble the initiation of chromosomal replication (for reviews, see references 18, 54, and 81). Controlled opening of the DNA duplex is required to permit entry of the DNA processing machinery. The task of remodeling the conjugative oriT is generally ascribed to two or three relaxosome auxiliary factors, of host and plasmid origin, which occupy specific DNA binding sites at this locus. Intrinsic to the relaxosome is also a site-and strand-specific DNA transesterase activity that breaks the phosphodiester backbone at nic (5). Upon cleavage, the transesterase enzyme (also called relaxase) forms a reversible phosphotyrosyl linkage to the 5Ј end of the DNA. Duplex unwinding initiating from this site produces the single-stranded T strand to be exported. A wealth of information is available supporting the importance of DNA sequence recognition and binding by relaxosome components at oriT to the transesterase reaction in vitro and for effective conjugative transfer (for reviews, see references 18, 54, and 81). On the other hand, the mechanisms controlling release of the 3Ј-OH generated at nic and the subsequent DNA unwind...

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A new trick for an old dog: TraY binding to a homopurine-homopyrimidine run attenuates DNA replication

Cited by 9 publications

References 59 publications

Mechanisms of Transcription-Replication Collisions in Bacteria

Mechanisms of Transcription-Replication Collisions in Bacteria

Replication Stalling at Friedreich's Ataxia (GAA)_n Repeats In Vivo

Plasmid R1 Conjugative DNA Processing Is Regulated at the Coupling Protein Interface

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A new trick for an old dog: TraY binding to a homopurine-homopyrimidine run attenuates DNA replication

Cited by 9 publications

References 59 publications

Mechanisms of Transcription-Replication Collisions in Bacteria

Mechanisms of Transcription-Replication Collisions in Bacteria

Replication Stalling at Friedreich's Ataxia (GAA)n Repeats In Vivo

Plasmid R1 Conjugative DNA Processing Is Regulated at the Coupling Protein Interface

Contact Info

Product

Resources

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Replication Stalling at Friedreich's Ataxia (GAA)_n Repeats In Vivo