[Show abstract][Hide abstract] ABSTRACT: Our laboratory specializes in directed protein evolution, i.e., evolution of proteins under defined selective pressures in the laboratory. Our target genes are encoded in ColE1 plasmids to facilitate the generation of libraries in vivo. We have observed that when random mutations are not restricted to the coding sequence of the target genes, directed evolution results in a strong positive selection of plasmid origin of replication (ori) mutations. Surprisingly, this is true even during evolution of new biochemical activities, when the activity that is being selected was not originally present. The selected plasmid ori mutations are diverse and produce a range of plasmid copy numbers, suggesting a complex interplay between ori and coding mutations rather than a simple enhancement of level of expression of the target gene. Thus, plasmid dosage may contribute significantly to evolution by fine-tuning levels of activity. Here, we present examples illustrating these observations as well as our methods for efficient quantification of plasmid copy number.
Full-text · Article · Jan 2012 · Methods in molecular biology (Clifton, N.J.)
[Show abstract][Hide abstract] ABSTRACT: DNA polymerase I (pol I) processes RNA primers during lagging-strand synthesis and fills small gaps during DNA repair reactions.
However, it is unclear how pol I and pol III work together during replication and repair or how extensive pol I processing
of Okazaki fragments is in vivo. Here, we address these questions by analyzing pol I mutations generated through error-prone replication of ColE1 plasmids.
The data were obtained by direct sequencing, allowing an accurate determination of the mutation spectrum and distribution.
Pol I’s mutational footprint suggests: (i) during leading-strand replication pol I is gradually replaced by pol III over at
least 1.3 kb; (ii) pol I processing of Okazaki fragments is limited to ∼20 nt and (iii) the size of Okazaki fragments is short
(∼250 nt). While based on ColE1 plasmid replication, our findings are likely relevant to other pol I replicative processes
such as chromosomal replication and DNA repair, which differ from ColE1 replication mostly at the recruitment steps. This
mutation footprinting approach should help establish the role of other prokaryotic or eukaryotic polymerases in vivo, and provides a tool to investigate how sequence topology, DNA damage, or interactions with protein partners may affect the
function of individual DNA polymerases.
Full-text · Article · May 2011 · Nucleic Acids Research