Everybody loves science!
I keep getting asked about what I'm studying this summer, so I thought I'd post my project proposal. It should be fairly readable, as that was one of the requirements for the fellowship application (the reason I wrote this at all). Let me know, though, because if it sounds all right, I will probably poach part of it for the introduction section of my senior thesis. If you have any questions, please ask them. I want it to be clear.
Recombination between repetitive extragenic palindrome sequences in Escherichia coli single-strand exonuclease mutants
The study of molecular genetics aims at understanding the method by which genes control a cell’s activities. Replication of these genes results in spontaneous mutation, which the cell attempts to fix via various repair mechanisms. Genetic repair is dependent on exonucleases; without them, genes quickly develop numerous serious mutations and are increasingly likely to recombine at illegitimate locations. Rearrangement often occurs at homeologous repeated segments along the genome. Studying recombination between these repeated segments will lead to better understanding of the processes of genetic replication, repair, and recombination.
Maintenance of its genetic code is vital for the survival of an organism and its progeny. During replication, however, genes often develop mutations due to a variety of factors both inside and outside the cell, including oxidation, radiation, chemical mutagens, and natural polymerase errors. Many of these genetic mistakes can be fixed via processes like methyl-directed mismatch repair (MMR), during which a helicase unwinds the DNA, a nuclease removes the erroneous section, and a polymerase fills the strand back in with the correct nucleotides. Repair processes such as MMR preserve genomic integrity, but they require nucleases to do so. Nucleases can either remove bases from the middle of a strand of DNA (endonucleases), or from the end (exonucleases). Both endo- and exo- nucleases can attack DNA in the 3’ direction, 5’ direction, or both directions, depending on the specificity of the nuclease.
The Lab has been studying the roles of exonucleases in the Escherichia coli bacterium, specifically those that destroy single-strand DNA (ssDNA). These roles include recombination, DNA repair, and mutation avoidance. Recent research has established that RecJ (a 5’-3’ exonuclease), ExoI (3’-5’), ExoVII (either direction), and ExoX (3’-5’) all take part in MMR, and that E. coli can use any one of them to correct its mutations in this way. Deleting the genes that code for all four of these exonucleases, though, severely reduces viability in E. coli. These single-strand exonuclease (ssExo-) mutants cannot grow at all in low temperatures and mutate rapidly in high temperatures in order to suppress their ssExo- genotype1.
The Lab also found that ssExo- mutants "appear to be especially prone to rearrangements at short sequence homologies" along the chromosome, where a particular sequence of bases is repeated either perfectly (homologous sections) or with a very similar pattern (homeologous sections)2. One of these sequences is the repetitive extragenic palindrome (REP) or palindromic unit (PU), a 40 base pair element that is repeated at least 500 times throughout the E. coli chromosome. These REP sequences are often found in clusters, with one REP sequence directly following another along the bacterial chromosome3. REP clusters, called bacterial interspersed mosaic elements (BIME), are always found in sections of E. coli operons that are transcribed but not translated, either between sections that code for genes or at the end of a set of genes4.
With so many places of rearrangement on the chromosome, the Lab hypothesizes that without single-strand exonucleases to help prevent rearrangements, E. coli recombines at REP loci until the genome is too scrambled to support life2. SsExo- strains are already prone to transition, deletion, and frameshift mutations, due to the inhibition of MMR and other repair functions, and the addition of a high likelihood of chromosomal rearrangement may push it past the threshold of viability. The four exonucleases also appear to have a role in the stabilization of repeated sections of DNA, preventing genomic slippage during replication2. In order to study the responsibilities of these exonucleases in preventing recombination at short sequence homologies, the Lab will assay for low-homology rearrangements. The expectation is that repair processes taking place in ssExo- mutants leaves bits of non-degraded ssDNA, which then recombines with the chromosomal DNA2.
The work I would like to do this summer involves rearrangements and BIME sequences. REP patterns are normally locationally stable within the genome; as such, they can be used to fingerprint strains or to study recombination. BIME are hotspots for recombination because their palindromic sequences can form hairpins in ssDNA and in RNA. A mutation in one side of the hairpin often leads to a mutation on the other side so that the strand can maintain its looped structure2, 3, 4. Also, a REP sequence may recombine with a second, inverted REP sequence, resulting in an inversion of the DNA between the two REP sequences. Other possible events during recombination include the deletion of the central portion entirely, if that section is excised when two directly repeated REP sequences recombine, or the addition of a second copy of the central section of DNA, should directly repeated REP sequences on different chromosomes recombine5.
By culturing ssExo- mutant strains and scanning for rearrangements with Southern blots, I can look for changes in the pattern of REP elements as compared to the wild-type. MMR and ssExo- mutants should show a high number of REP inversions, deletions, and duplications, which will be visible in the banding pattern on a Southern blot. Using restriction enzymes to cut the E. coli DNA into pieces, I should also be able to find gene conversion between the REP elements. If there are not enough rearrangements to be visible by blotting, I can use specific primers to amplify the recombinant sections via the polymerase chain reaction (PCR). This should help to detect less common rearrangements.
The PCR process may also be used to find otherwise undetectable inverted sequences. During PCR, DNA is denatured into single strands, primers bind to specific sections flanking the section of DNA to be copied, and polymerase attaches to the primers to begin replication. By setting up a segment of DNA in which two flanking sections point the same way on the original DNA, I can tell by the size of the PCR products whether the section between the flanking areas becomes inverted. If the segment is unaltered, amplification will proceed as usual at both loci. If inversion occurs, then the primers will point in opposite directions and the amplification process will result in small bits of DNA, for the two flanking regions will be close together and the polymerases will be competing to work on the same stretch of DNA. After analyzing these various mutations, I can compare MMR mutants with exonuclease mutants to see if the latter builds upon the high level of rearrangement seen in MMR mutants, and what other effect the two types of mutation have together.
This project will add to scientific knowledge about how DNA recombination, repair, and mutation avoidance work, especially as pertaining to REP sequences. At the moment, REP sequences are not well understood, despite the fact that they take up about 1% of the E. coli genome3, 4. They seem to be involved with chromosomal rearrangements and are implicated in the structure and evolution of genomes3, but their effect on the regulation of relative gene expression within operons does not account for much of the differences in expression, as once hypothesized4. REP sequences may still have a role in gene expression and chromosome stability, in addition to the proposed role in the facilitation of DNA rearrangement. It is my hope that my research will help to explain why there are so many BIME within the E. coli genome.
1. Viswanathan, M., V. Burdett, C. Baitinger, P. Modrich, and S.T. Lovett. 2001. Redundant exonuclease involvement in Escherichia coli methyl-directed mismatch repair. J. Biol. Chem. 276:31053-8.
2. Lovett, S. T. 2002. Application for a grant toward the study of nucleases in E. coli and S. cerevisiae.
3. Lupski, James R., and George M. Weinstock. 1992. Short, interspersed repetitive DNA sequences in prokaryotic genomes. J. Bacteriology. 174:4525-9.
4. Stern, M. J., G. F-L. Ames, N. H. Smith, E. C. Robinson, and C. F. Higgins. 1984. Repetitive extragenic palindromic sequences: a major component of the bacterial genome. Cell. 37:1015-26.
5. Bachellier, S., E. Gilson, M. Hofnung, and C. W. Hill. "Repeated Sequences." 2012-40.
One note: Writing scientific papers/laboratory reports/articles/proposals/whatever always drives me batty. You're supposed to use excessive passive voice in sciencey things, but a few of my neurons die every time I do it. Argh.
come back and take you home
I could not stop, that you now know
home, home, where I wanted to go
Recombination between repetitive extragenic palindrome sequences in Escherichia coli single-strand exonuclease mutants
The study of molecular genetics aims at understanding the method by which genes control a cell’s activities. Replication of these genes results in spontaneous mutation, which the cell attempts to fix via various repair mechanisms. Genetic repair is dependent on exonucleases; without them, genes quickly develop numerous serious mutations and are increasingly likely to recombine at illegitimate locations. Rearrangement often occurs at homeologous repeated segments along the genome. Studying recombination between these repeated segments will lead to better understanding of the processes of genetic replication, repair, and recombination.
Maintenance of its genetic code is vital for the survival of an organism and its progeny. During replication, however, genes often develop mutations due to a variety of factors both inside and outside the cell, including oxidation, radiation, chemical mutagens, and natural polymerase errors. Many of these genetic mistakes can be fixed via processes like methyl-directed mismatch repair (MMR), during which a helicase unwinds the DNA, a nuclease removes the erroneous section, and a polymerase fills the strand back in with the correct nucleotides. Repair processes such as MMR preserve genomic integrity, but they require nucleases to do so. Nucleases can either remove bases from the middle of a strand of DNA (endonucleases), or from the end (exonucleases). Both endo- and exo- nucleases can attack DNA in the 3’ direction, 5’ direction, or both directions, depending on the specificity of the nuclease.
The Lab has been studying the roles of exonucleases in the Escherichia coli bacterium, specifically those that destroy single-strand DNA (ssDNA). These roles include recombination, DNA repair, and mutation avoidance. Recent research has established that RecJ (a 5’-3’ exonuclease), ExoI (3’-5’), ExoVII (either direction), and ExoX (3’-5’) all take part in MMR, and that E. coli can use any one of them to correct its mutations in this way. Deleting the genes that code for all four of these exonucleases, though, severely reduces viability in E. coli. These single-strand exonuclease (ssExo-) mutants cannot grow at all in low temperatures and mutate rapidly in high temperatures in order to suppress their ssExo- genotype1.
The Lab also found that ssExo- mutants "appear to be especially prone to rearrangements at short sequence homologies" along the chromosome, where a particular sequence of bases is repeated either perfectly (homologous sections) or with a very similar pattern (homeologous sections)2. One of these sequences is the repetitive extragenic palindrome (REP) or palindromic unit (PU), a 40 base pair element that is repeated at least 500 times throughout the E. coli chromosome. These REP sequences are often found in clusters, with one REP sequence directly following another along the bacterial chromosome3. REP clusters, called bacterial interspersed mosaic elements (BIME), are always found in sections of E. coli operons that are transcribed but not translated, either between sections that code for genes or at the end of a set of genes4.
With so many places of rearrangement on the chromosome, the Lab hypothesizes that without single-strand exonucleases to help prevent rearrangements, E. coli recombines at REP loci until the genome is too scrambled to support life2. SsExo- strains are already prone to transition, deletion, and frameshift mutations, due to the inhibition of MMR and other repair functions, and the addition of a high likelihood of chromosomal rearrangement may push it past the threshold of viability. The four exonucleases also appear to have a role in the stabilization of repeated sections of DNA, preventing genomic slippage during replication2. In order to study the responsibilities of these exonucleases in preventing recombination at short sequence homologies, the Lab will assay for low-homology rearrangements. The expectation is that repair processes taking place in ssExo- mutants leaves bits of non-degraded ssDNA, which then recombines with the chromosomal DNA2.
The work I would like to do this summer involves rearrangements and BIME sequences. REP patterns are normally locationally stable within the genome; as such, they can be used to fingerprint strains or to study recombination. BIME are hotspots for recombination because their palindromic sequences can form hairpins in ssDNA and in RNA. A mutation in one side of the hairpin often leads to a mutation on the other side so that the strand can maintain its looped structure2, 3, 4. Also, a REP sequence may recombine with a second, inverted REP sequence, resulting in an inversion of the DNA between the two REP sequences. Other possible events during recombination include the deletion of the central portion entirely, if that section is excised when two directly repeated REP sequences recombine, or the addition of a second copy of the central section of DNA, should directly repeated REP sequences on different chromosomes recombine5.
By culturing ssExo- mutant strains and scanning for rearrangements with Southern blots, I can look for changes in the pattern of REP elements as compared to the wild-type. MMR and ssExo- mutants should show a high number of REP inversions, deletions, and duplications, which will be visible in the banding pattern on a Southern blot. Using restriction enzymes to cut the E. coli DNA into pieces, I should also be able to find gene conversion between the REP elements. If there are not enough rearrangements to be visible by blotting, I can use specific primers to amplify the recombinant sections via the polymerase chain reaction (PCR). This should help to detect less common rearrangements.
The PCR process may also be used to find otherwise undetectable inverted sequences. During PCR, DNA is denatured into single strands, primers bind to specific sections flanking the section of DNA to be copied, and polymerase attaches to the primers to begin replication. By setting up a segment of DNA in which two flanking sections point the same way on the original DNA, I can tell by the size of the PCR products whether the section between the flanking areas becomes inverted. If the segment is unaltered, amplification will proceed as usual at both loci. If inversion occurs, then the primers will point in opposite directions and the amplification process will result in small bits of DNA, for the two flanking regions will be close together and the polymerases will be competing to work on the same stretch of DNA. After analyzing these various mutations, I can compare MMR mutants with exonuclease mutants to see if the latter builds upon the high level of rearrangement seen in MMR mutants, and what other effect the two types of mutation have together.
This project will add to scientific knowledge about how DNA recombination, repair, and mutation avoidance work, especially as pertaining to REP sequences. At the moment, REP sequences are not well understood, despite the fact that they take up about 1% of the E. coli genome3, 4. They seem to be involved with chromosomal rearrangements and are implicated in the structure and evolution of genomes3, but their effect on the regulation of relative gene expression within operons does not account for much of the differences in expression, as once hypothesized4. REP sequences may still have a role in gene expression and chromosome stability, in addition to the proposed role in the facilitation of DNA rearrangement. It is my hope that my research will help to explain why there are so many BIME within the E. coli genome.
1. Viswanathan, M., V. Burdett, C. Baitinger, P. Modrich, and S.T. Lovett. 2001. Redundant exonuclease involvement in Escherichia coli methyl-directed mismatch repair. J. Biol. Chem. 276:31053-8.
2. Lovett, S. T. 2002. Application for a grant toward the study of nucleases in E. coli and S. cerevisiae.
3. Lupski, James R., and George M. Weinstock. 1992. Short, interspersed repetitive DNA sequences in prokaryotic genomes. J. Bacteriology. 174:4525-9.
4. Stern, M. J., G. F-L. Ames, N. H. Smith, E. C. Robinson, and C. F. Higgins. 1984. Repetitive extragenic palindromic sequences: a major component of the bacterial genome. Cell. 37:1015-26.
5. Bachellier, S., E. Gilson, M. Hofnung, and C. W. Hill. "Repeated Sequences." 2012-40.
One note: Writing scientific papers/laboratory reports/articles/proposals/whatever always drives me batty. You're supposed to use excessive passive voice in sciencey things, but a few of my neurons die every time I do it. Argh.
come back and take you home
I could not stop, that you now know
home, home, where I wanted to go