Affiliation:
1. Program in Biological and Biomedical Sciences, Graduate School of Arts and Sciences, Harvard Medical School, Boston, Massachusetts, USA
2. Division of Infectious Diseases, Brigham and Women's Hospital, Boston, Massachusetts, USA
3. Howard Hughes Medical Institute, Boston, Massachusetts, USA
Abstract
ABSTRACT
Vibrionaceae
family members are interesting models for studying DNA replication initiation, as they contain two circular chromosomes. Chromosome II (chrII) replication is governed by two evolutionarily unique yet highly conserved elements, the origin DNA sequence
oriCII
and the initiator protein RctB. The minimum functional region of
oriCII
,
oriCII-min
, contains multiple elements that are bound by RctB
in vitro
, but little is known about the specific requirements for individual elements during
oriCII
initiation. We utilized undirected and site-specific mutagenesis to investigate the functionality of mutant forms of
oriCII-min
and assessed binding to various mutant forms by RctB. Our analyses showed that deletions, point mutations, and changes in RctB target site spacing or methylation all impaired
oriCII-min
-based replication. RctB displayed a reduced affinity for most of the low-efficacy origins tested, although its characteristic cooperative binding was generally maintained. Mutations that removed or altered the relative positions of origin components other than RctB binding sites (e.g., AT-rich sequence, DnaA target site) also abolished replicative capacity. Comprehensive mutagenesis and deep-sequencing-based screening (OriSeq) allowed the identification of a previously uncharacterized methylated domain in
oriCII
that is required for origin function. Together, our results reveal the remarkable evolutionary honing of
oriCII
and provide new insight into the complex interplay between RctB and
oriCII
.
IMPORTANCE
The genome of the enteric pathogen
Vibrio cholerae
consists of two chromosomes. While the chromosome I replication origin and its cognate replication initiator protein resemble those of
Escherichia coli
, the factors responsible for chromosome II replication initiation display no similarity to any other known initiation systems. Here, to enhance our understanding of how this DNA sequence,
oriCII
, and its initiator protein, RctB, function, we used both targeted mutagenesis and a new random-mutagenesis approach (OriSeq) to finely map the
oriCII
structural features and sequences required for RctB-mediated DNA replication. Collectively, our findings reveal the extraordinary evolutionary honing of the architecture and motifs that constitute
oriCII
and reveal a new role for methylation in
oriCII
-based replication. Finally, our findings suggest that the OriSeq approach is likely to be widely applicable for defining critical bases in
cis
-acting sequences.
Publisher
American Society for Microbiology
Cited by
20 articles.
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