Andrei Kuzminov
Associate Professor of Microbiology
Education
M.A. (Biochemistry) University of Novosibirsk, Russia, 1985
Ph.D. (Molecular Biology) Institute of Cytology and Genetics, Novosibirsk, Russia, 1990
Postdoctoral (Molecular Genetics) Institute of Molecular Biology, University of Oregon, 1991-2000
Teaching Interests
Chromosomal fragmentation: mechanisms, repair, avoidance
Figure 1. The idea of replication fork collapse at a single-strand interruption in the template DNA and subsequent RecA-catalyzed strand exchange and replication fork restart. This is one of the scenarios of "replication fork failure" that we are investigating.
Postdoctoral Research Opportunity
Chromosomal fragmentation is a result of double-strand DNA breaks. If left unrepaired, such double-strand breaks paralyze the chromosomal metabolism, blocking DNA replication, interfering with chromosomal segregation and leading to a direct loss of genetic material due to DNA degradation. There is a variety of fascinating mechanisms the organisms employ to repair double-strand DNA breaks. Still, this repair is complex and costly; besides, elevated chromosomal fragmentation still leads to genetic instability.
Therefore, it is not surprising that the cells prefer to avoid chromosomal fragmentation altogether. As we have recently discovered, cells employ a variety of clever ways to do it. We found mutants in Escherichia coli that are defective in this avoidance of chromosomal fragmentation and become dependent on double-strand break repair. Analogous mutants in higher eukaryotes, via elevated genetic instability, should be predisposed to cancer.
Another major breakthrough that this laboratory is spearheading is the realization that the mechanisms underlying chromosomal fragmentation have little to do with spontaneous direct double-strand breaks and are all linked to the newly-discovered phenomenon of replication fork failure. We are just beginning to grasp the variety of ways it is possible to break duplex DNA during replication. Several proposed mechanisms of replication-dependent chromosomal fragmentation are being tested and more are suspected.
We employ two major approaches to study the mechanisms of chromosomal fragmentation, as well as its repair and avoidance. Our genomic approach includes insertional mutagenesis, screens for synthetic lethals and selections for suppressors of synthetic lethals. The interesting mutants that we isolate are then subjected to a variety of physical methods of analysis, including pulsed-field gel electrophoresis, differential labeling, marker frequency analysis, 2D-gel electrophoresis, sucrose gradient centrifugation, 2D TLC.
The current projects in the laboratory include
- Patterns of DNA fragmentation around the chromosome: the gradient, the polarity and the role of the replication origin.
- The mechanisms of chromosomal fragmentation caused by non-canonical DNA precursors like dUTP and dITP. Why is it important to keep your DNA free of base-analogs even though they are non-mutagenic?
- The mechanisms of chromosomal fragmentation due to overinitiation of the chromosomal replication. What is the role of the proper nucleoid administration in avoidance of chromosomal lesions?
- The mechanism of synthesis of the leading strand during DNA replication. Is replication fork stalled at one-strand DNA lesions or does it continue past them, forming daughter-strand gaps?
- The pathways of synthesis of the non-canonical DNA precursor dITP. Widespread moonlighting by enzymes of the nucleotide metabolism?
- Cellular consequences of the inability to intercept non-canonical DNA precursors. What to do if the DNA precursor pools are invaded by modified nucleotides?
- The lethal imbalance of the DNA precursor pools: the enigmatic thymineless death
Figure 2.The color screen for recA-dependent mutants. This is our major approach to identify chromosomal fragmentation-avoidance strategies. Mutants with a chromosomal-avoidance pathway inactivated form non-sectoring, solidly-colored colonies. Two such mutants, dut and rdgB, are shown in the insert.
Figure 3.RecA-dependent mutants reveal chromosomal fragmentation when combined with a recBC(Ts) defect. We use pulsed-field gel electrophoresis to detect chromosomal fragmentation and then to inquire into its mechanisms. Intact chromosomes stay in the wells, while fragmented chromosomes form a characteristic smear in the gel. The three rightmost strains fragment their chromosomes at 37°C.
Representative Publications
Kouzminova, E.A. and Kuzminov, A. 2008. Patterns of chromosomal fragmentation due to uracil-DNA incorporation reveal a novel mechanism of replication-dependent double-strand breaks. Molecular Microbiology, in press.
Rotman, E. and Kuzminov, A. 2007. The mutT defect does not elevate chromosomal fragmentation in Escherichia coli because of the surprisingly low levels of MutM/MutY-recognized DNA modifications. J. Bacteriol., 189:6976–88. [Abstract]
Budke, B. and Kuzminov, A. 2006. Hypoxanthine incorporation is non-mutagenic in Escherichia coli. J. Bacteriol., 188:6553–60. [Abstract]
Amado, L. and Kuzminov, A. 2006. The replication intermediates in Escherichia coli are not the product of DNA processing or uracil excision. J. Biol. Chem., 281:22635–46. [Abstract]
Lukas, L. and Kuzminov, A. 2006. Chromosomal fragmentation is the major consequence of the rdgB defect in Escherichia coli. Genetics, 172:1359–62. [Abstract]
Kouzminova, E.A. and Kuzminov, A. 2006. Fragmentation of replicating chromosomes triggered by uracil in DNA. J. Mol. Biol., 355:20–33. [Abstract]
Kouzminova, E.A., Rotman, E, Macomber, L., Zhang, J., and Kuzminov, A. 2004. RecA-dependent mutants in Escherichia coli reveal strategies to avoid chromosomal fragmentation. Proc. Natl. Acad. Sci. USA, 101:16262–7. [Abstract]