Transposase interaction with the β sliding clamp: effects on insertion sequence proliferation and transposition rate

In the mid 1970s, Miroslav Radman and Evelyn Witkin proposed that Escherichia coli must encode a specialized error-prone DNA polymerase (pol) to account for the 100-fold increase in mutations accompanying induction of the SOS regulon. By the late 1980s, genetic studies showed that SOS mutagenesis required the presence of two “UV mutagenesis” genes, umuC and umuD, along with recA. Guided by the genetics, decades of biochemical studies have defined the predicted error-prone DNA polymerase as an activated complex of these three gene products, assembled as a mutasome, pol V Mut = UmuD’2C-RecA-ATP. Here, we explore the role of the β-sliding processivity clamp on the efficiency of pol V Mut-catalyzed DNA synthesis on undamaged DNA and during translesion DNA synthesis (TLS). Primer elongation efficiencies and TLS were strongly enhanced in the presence of β. The results suggest that β may have two stabilizing roles: its canonical role in tethering the pol at a primer-3’-terminus, and a possible second role in inhibiting pol V Mut’s ATPase to reduce the rate of mutasome-DNA dissociation. The identification of umuC, umuD, and recA homologs in numerous strains of pathogenic bacteria and plasmids will ensure the long and productive continuation of the genetic and biochemical journey initiated by Radman and Witkin.

The Functional Consequences of the Novel Ribosomal Pausing Site in SARS-CoV-2 Spike Glycoprotein RNA

International Journal of Molecular Sciences ◽

10.3390/ijms22126490 ◽

2021 ◽

Vol 22 (12) ◽

pp. 6490

Author(s):

Olga A. Postnikova ◽

Sheetal Uppal ◽

Weiliang Huang ◽

Maureen A. Kane ◽

Rafael Villasmil ◽

...

Keyword(s):

Amino Acid ◽

Insertion Sequence ◽

Experimental Studies ◽

Spike Protein ◽

Spike Glycoprotein ◽

Furin Cleavage ◽

New Host ◽

S Protein ◽

Furin Cleavage Site ◽

Ribosome Pausing

The SARS-CoV-2 Spike glycoprotein (S protein) acquired a unique new 4 amino acid -PRRA- insertion sequence at amino acid residues (aa) 681–684 that forms a new furin cleavage site in S protein as well as several new glycosylation sites. We studied various statistical properties of the -PRRA- insertion at the RNA level (CCUCGGCGGGCA). The nucleotide composition and codon usage of this sequence are different from the rest of the SARS-CoV-2 genome. One of such features is two tandem CGG codons, although the CGG codon is the rarest codon in the SARS-CoV-2 genome. This suggests that the insertion sequence could cause ribosome pausing as the result of these rare codons. Due to population variants, the Nextstrain divergence measure of the CCU codon is extremely large. We cannot exclude that this divergence might affect host immune responses/effectiveness of SARS-CoV-2 vaccines, possibilities awaiting further investigation. Our experimental studies show that the expression level of original RNA sequence “wildtype” spike protein is much lower than for codon-optimized spike protein in all studied cell lines. Interestingly, the original spike sequence produces a higher titer of pseudoviral particles and a higher level of infection. Further mutagenesis experiments suggest that this dual-effect insert, comprised of a combination of overlapping translation pausing and furin sites, has allowed SARS-CoV-2 to infect its new host (human) more readily. This underlines the importance of ribosome pausing to allow efficient regulation of protein expression and also of cotranslational subdomain folding.

Eukaryotic clamp loaders and unloaders in the maintenance of genome stability

Experimental & Molecular Medicine ◽

10.1038/s12276-020-00533-3 ◽

2020 ◽

Vol 52 (12) ◽

pp. 1948-1958

Author(s):

Kyoo-young Lee ◽

Su Hyung Park

Keyword(s):

Dna Damage ◽

Genomic Instability ◽

Genome Stability ◽

Critical Role ◽

Nuclear Antigen ◽

Checkpoint Activation ◽

Sliding Clamp ◽

Clamp Loaders ◽

Factor C

AbstractEukaryotic sliding clamp proliferating cell nuclear antigen (PCNA) plays a critical role as a processivity factor for DNA polymerases and as a binding and acting platform for many proteins. The ring-shaped PCNA homotrimer and the DNA damage checkpoint clamp 9-1-1 are loaded onto DNA by clamp loaders. PCNA can be loaded by the pentameric replication factor C (RFC) complex and the CTF18-RFC-like complex (RLC) in vitro. In cells, each complex loads PCNA for different purposes; RFC-loaded PCNA is essential for DNA replication, while CTF18-RLC-loaded PCNA participates in cohesion establishment and checkpoint activation. After completing its tasks, PCNA is unloaded by ATAD5 (Elg1 in yeast)-RLC. The 9-1-1 clamp is loaded at DNA damage sites by RAD17 (Rad24 in yeast)-RLC. All five RFC complex components, but none of the three large subunits of RLC, CTF18, ATAD5, or RAD17, are essential for cell survival; however, deficiency of the three RLC proteins leads to genomic instability. In this review, we describe recent findings that contribute to the understanding of the basic roles of the RFC complex and RLCs and how genomic instability due to deficiency of the three RLCs is linked to the molecular and cellular activity of RLC, particularly focusing on ATAD5 (Elg1).

Distribution of the ermG Gene among Bacterial Isolates from Porcine Intestinal Contents

Applied and Environmental Microbiology ◽

10.1128/aem.71.8.4930-4934.2005 ◽

2005 ◽

Vol 71 (8) ◽

pp. 4930-4934 ◽

Cited By ~ 17

Author(s):

Yanping Wang ◽

Gui-Rong Wang ◽

Nadja B. Shoemaker ◽

Terence R. Whitehead ◽

Abigail A. Salyers

Keyword(s):

Insertion Sequence ◽

Bacillus Sphaericus ◽

Gram Positive ◽

Sequence Element ◽

Conjugative Transposon ◽

Gram Positive Bacteria ◽

First Finding ◽

Intestinal Contents ◽

Porcine Feces ◽

Insertion Sequence Element

ABSTRACT The ermG gene was first found in the soil bacterium Bacillus sphaericus. More recently, it was found in several human intestinal Bacteroides species. We report here the first finding of ermG genes in gram-positive bacteria isolated from porcine feces and from under-barn manure pits used to store porcine wastes. The porcine ermG sequences were identical to the sequence of the B. sphaericus ermG gene except that six of the seven ermG-containing strains contained an insertion sequence element insertion in the C-terminal end of the gene. The porcine ermG genes were found in three different gram-positive genera, an indication that it is possible that the gene is being spread by horizontal gene transfer. A segment of a Bacteroides conjugative transposon that carries an ermG gene cross-hybridized with DNA from six of the seven porcine isolates, but the restriction patterns in the porcine strains were different from that of the Bacteroides conjugative transposon.