Biomedical subjects

Susanna M Lewis

Publications and source records attributed to Susanna M Lewis.

4 recordsLinked to original sources

PubMed2006

Palindromes and genomic stress fractures: bracing and repairing the damage.

DNA palindromes are a source of instability in eukaryotic genomes but remain under-investigated because they are difficult to study. Nonetheless, progress in the last year or so has begun to form a coherent picture of how DNA palindromes cause damage in eukaryotes and how this damage is opposed by cellular mechanisms. In yeast, the features of double strand DNA interruptions that appear at palindromic sites in vivo suggest that a resolvase-type activity creates the fractures by attacking a palindrome after it extrudes into a cruciform structure. Induction of DNA breaks in this fashion could be deterred through a Center-Break palindrome revision process as investigated in detail in mice. The MRX/MRN likely plays a pivotal role in prevention of palindrome-induced genome damage in eukaryotes.

Animals↗

PubMed2005

New approaches to the analysis of palindromic sequences from the human genome: evolution and polymorphism of an intronic site at the NF1 locus.

The nature of any long palindrome that might exist in the human genome is obscured by the instability of such sequences once cloned in Escherichia coli. We describe and validate a practical alternative to the analysis of naturally-occurring palindromes based upon cloning and propagation in Saccharomyces cerevisiae. With this approach we have investigated an intronic sequence in the human Neurofibromatosis 1 (NF1) locus that is represented by multiple conflicting versions in GenBank. We find that the site is highly polymorphic, exhibiting different degrees of palindromy in different individuals. A side-by-side comparison of the same plasmids in E.coli versus. S.cerevisiae demonstrated that the more palindromic alleles were inevitably corrupted upon cloning in E.coli, but could be propagated intact in yeast. The high quality sequence obtained from the yeast-based approach provides insight into the various mechanisms that destabilize a palindrome in E.coli, yeast and humans, into the diversification of a highly polymorphic site within the NF1 locus during primate evolution, and into the association between palindromy and chromosomal translocation.

Alleles↗

PubMed2005

The recombination difference between mouse kappa and lambda segments is mediated by a pair-wise regulation mechanism.

In mice, kappa light chains dominate over lambda in the immunoglobulin repertoire by as much as 20-fold. Although a major contributor to this difference is the recombination signal sequences (RSS), the mechanism by which RSS cause differential representation has not been determined. To elucidate the mechanism, we tested kappa and lambda RSS flanked by their natural 5' and 3' flanks in three systems that monitor V(D)J recombination. Using extra-chromosomal recombination substrates, we established that a kappa RSS and its flanks support six- to nine-fold higher levels of recombination than a lambda counterpart. In vitro cleavage assays with these same sequences demonstrated that single cleavage at individual kappa or lambda RSS (plus flanks) occurs with comparable frequencies, but that a pair of kappa RSS (plus flanks) support significantly higher levels of double cleavage than a pair of lambda RSS (plus flanks). Using EMSA with double stranded oligonucleotides containing the same kappa or lambda RSS and their respective flanks, we examined RAG/DNA complex formation. We report that, surprisingly, RAG-1/2 form only modestly higher levels of complexes on individual 12 and 23 kappa RSS (plus natural flanks) as compared to their lambda counterparts. We conclude that the overuse of kappa compared to lambda segments cannot be accounted for by differences in RAG-1/2 binding nor by cleavage at individual RSS but rather could be accounted for by enhanced pair-wise cleavage of kappa RSS by RAG-1/2. Based on the data presented, we suggest that the biased usage of light chain segments is imposed at the level of synaptic RSS pairs.

Animals↗

PubMed2003

Rapid, stabilizing palindrome rearrangements in somatic cells by the center-break mechanism.

DNA palindromes are associated with rearrangement in a variety of organisms. A unique opportunity to examine the impact of a long palindrome in mammals is afforded by the Line 78 strain of mice. Previously it was found that the transgene in Line 78 is likely to be palindromic and that the symmetry of the transgene was responsible for a high level of germ line instability. Here we prove that Line 78 mice harbor a true 15.4-kb palindrome, and through the establishment of cell lines from Line 78 mice we have shown that the palindrome rearranges at the impressive rate of about 0.5% per population doubling. The rearrangements observed to arise from rapid palindrome modification are consistent with a center-break mechanism where double-strand breaks, created through hairpin nicking of an extruded cruciform, are imprecisely rejoined, thus introducing deletions at the palindrome center. Significantly, palindrome rearrangements in somatic tissue culture cells almost completely mirrored the structures generated in vivo in the mouse germ line. The close correspondence between germ line and somatic events indicates the possibility that center-break modification of palindromes is an important mechanism for preventing mutation in both contexts. Permanent cell lines carrying a verified palindrome provide an essential tool for future mechanistic analyses into the consequences of palindromy in the mammalian genome.

Animals↗