Chromosomal Replication Complexity: A Novel DNA Metrics and Genome Instability Factor

Kuzminov, Andrei

doi:10.1371/journal.pgen.1006229

Cited by 19 publications

(19 citation statements)

References 114 publications

(145 reference statements)

Supporting

Mentioning

Contrasting

Order By: Relevance

“…Rather than by fork fusion events themselves, might the over-replication be caused by a cryptic origin that is normally suppressed, or by the increased occurrence of R-loops within the termination area, as recently suggested (Kuzminov, 2016)? While remaining a possibility, it is unlikely for a number of reasons.…”

Section: Making Sense Of the Replication Fork Trapmentioning

confidence: 98%

Too Much of a Good Thing: How Ectopic DNA Replication Affects Bacterial Replication Dynamics

et al. 2020

View full text Add to dashboard Cite

Section: Making Sense Of the Replication Fork Trapmentioning

confidence: 98%

Too Much of a Good Thing: How Ectopic DNA Replication Affects Bacterial Replication Dynamics

et al. 2020

View full text Add to dashboard Cite

“…16 dsDNA breaks lead to induction of the SOS response, which results in increased RecA (a rad51 homologue) based homologous recombination. 26 As a typical E. coli cell contains multiple chromosomes (from 1 to 8, depending on growth rate), 27 sister chromatids offer templates for recombination based repair of Cas9 induced cuts. Thus, as illustrated in Figure 2a, Cas9-mediated cell death takes place only if all copies of the chromosome are cleaved faster than they are repaired.…”

Section: Measuring "On Target" Cutting Rates Via An E Coli Survival mentioning

confidence: 99%

CRISPR/Cas “non-target” sites inhibit on-target cutting rates

Moreb¹,

Hutmacher²,

Lynch³

2020

Preprint

View full text Add to dashboard Cite

CRISPR/Cas systems have become ubiquitous for genome editing in eukaryotic as well as bacterial systems. Cas9 associated with a guide RNA (gRNA) searches DNA for a matching sequence (target site) next to a protospacer adjacent motif (PAM) and once found, cuts the DNA.The number of PAM sites in the genome are effectively a non-target pool of inhibitory substrates, competing with the target site for the Cas9/gRNA complex. We demonstrate that increasing the number of non-target sites for a given gRNA reduces on-target activity in a dose dependent manner. Furthermore, we show that the use of Cas9 mutants with increased PAM specificity towards a smaller subset of PAMs (or smaller pool of competitive substrates) improves cutting rates. Decreasing the non-target pool by increasing PAM specificity provides a path towards improving on-target activity for slower high fidelity Cas9 variants. These results demonstrate the importance of competitive non-target sites on Cas9 activity and, in part, may help to explain sequence and context dependent activities of gRNAs. Engineering improved PAM specificity to reduce the competitive non-target pool offers an alternative strategy to engineer Cas9 variants with increased specificity and maintained on-target activity.

show abstract

“…Chromosomal replication complexity (CRC) represents the ratio of the copy number of the most replicated region in the chromosome to the copy number of the unreplicated regions (25,26). In bacterial chromosomes, the CRC is simply expressed as the origin/terminus (ori/ter) ratio.…”

mentioning

confidence: 99%

Thymineless Death in Escherichia coli Is Unaffected by Chromosomal Replication Complexity

Khan

Kuzminov

2019

J Bacteriol

Self Cite

View full text Add to dashboard Cite

Thymineless death (TLD) is a rapid loss of viability of unclear mechanism in cultures of thyA mutants starved for thymine/thymidine (T starvation). It is accepted that T starvation repeatedly breaks replication forks, while recombinational repair restores them, but when the resulting futile breakage-repair cycle affects the small replication bubbles at oriC, the origin is degraded, killing the cell. Indeed, cells with increased chromosomal replication complexity (CRC), expressed as an elevated origin/terminus (ori/ter) ratio, die more extensively during TLD. Here we tested this logic by elevating the CRC in Escherichia coli thyA mutants before T starvation, anticipating exaggerated TLD. Unexpectedly, TLD remained unaffected by a CRC increase to either the natural limit (ori/ter ratio, ϳ6) or the functional limit (ori/ter ratio, ϳ16). Moreover, when we forced the CRC over the functional limit (ori/ter ratio, ϳ30), TLD lessened. Thus, prior overinitiation does not sensitize cells to TLD. In contradiction with the published results, even blocking new replication initiations by the dnaA(Ts) defect at 42°C fails to prevent TLD. Using the thyA dnaA(Ts) mutant in a new T starvation protocol that excludes new initiations, we show that at 42°C, the same degree of TLD still occurs when chromosomes are demonstrably nonreplicating. Remarkably, 80% of the chromosomal DNA in these nonreplicating T-starved cells is still lost, by an unclear mechanism. IMPORTANCE Thymineless death kills cells of any type and is used in anticancer and antimicrobial treatments. We tested the idea that the more replication forks there are in the chromosome during growth, the more extensive the resulting thymineless death. We varied the number of replication forks in the Escherichia coli chromosome, as measured by the origin-to-terminus ratio, ranging it from the normal 2 to 60, and even completely eliminated replication forks in the nonreplicating chromosomes (ori/ter ratio ϭ 1). Unexpectedly, we found that thymineless death is unaffected by the intensity of replication or by its complete absence; we also found that even nonreplicating chromosomes still disappear during thymine starvation. We conclude that thymineless death can kill E. coli independently of chromosomal replication.

show abstract

Chromosomal Replication Complexity: A Novel DNA Metrics and Genome Instability Factor

Cited by 19 publications

References 114 publications

Too Much of a Good Thing: How Ectopic DNA Replication Affects Bacterial Replication Dynamics

Too Much of a Good Thing: How Ectopic DNA Replication Affects Bacterial Replication Dynamics

CRISPR/Cas “non-target” sites inhibit on-target cutting rates

Thymineless Death in Escherichia coli Is Unaffected by Chromosomal Replication Complexity

Contact Info

Product

Resources

About