Office: (217) 265-0329
Lab: (217) 265-0852
Mail to: B103 CLSL
Professor of Microbiology
DNA Biology, Genetics, Genomics, Host-Pathogen Interactions, Microbial Physiology
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
Mechanisms of DNA replication stress and genome instability
Genome instability is of critical concern in human health, as it underlies the process of aging, diseases like cancer and neurodegenerative disorders, and the emergence of drug-resistant pathogens. At the same time, controlling genome instability and targeting it against the undesirable cells is a prominent feature of many host-pathogen interactions and is at the basis of efficient anti-cancer, anti-viral and anti-microbial treatments. The genome instability research was transformed by realization, which we spearheaded, that the focus of the multiple instability mechanisms are replication forks. In this laboratory, we study DNA replication stress causing abnormal behavior of replication forks, and how this leads to various kinds of chromosomal lesions, which are direct precursors of any genetic instability, resulting in cancerous transformation.
Since the mechanisms of genome instability are as basic as the mechanisms of DNA replication itself and therefore are shared between all domains of Life, to study them we employ the most tractable experimental system, which is Escherichia coli, utilizing two major approaches. Our powerful 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, LC-MS, etc. We also use the same physical approaches to study chromosomal lesions induced by various DNA-damaging treatments, with the overarching goal to understand how DNA damage kills.
Endogenous chromosomal fragmentation, cancer predisposition and cancer-avoidance
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. Organisms employ fascinating mechanisms to repair double-strand DNA breaks. Recombinational repair is the most complex one, offering precise restoration of the genetic information. Harnessing the mechanisms of recombinational repair will open the door to precise gene targeting and gene therapy. However, such a precise repair of fragmented chromosomes is complex and costly; besides, elevated chromosomal fragmentation, even if completely repairable, still leads to genome instability and cancer.
Therefore, it is not surprising that the cells prefer to avoid chromosomal fragmentation altogether. As we have recently demonstrated, cells employ a variety of clever strategies to do this. We found mutants that are defective in this avoidance of chromosomal fragmentation and, therefore, dependent on double-strand break repair. Analogous mutations in higher eukaryotes cause elevated genetic instability and predisposition to cancer. Thus, our mechanistic studies, which are impossible to perform in the cells of higher eukaryotes, provide critical insights into the mechanisms of early carcinogenesis, as well as of cancer-avoidance.
Another major breakthrough that this laboratory is spearheading is the realization that the mechanisms underlying chromosomal fragmentation have little to do with direct double-strand breaks and are all linked to the novel phenomena associated with DNA replication stress and replication fork failure. We are beginning to characterize the variety of ways to break duplex DNA during replication. Several proposed mechanisms of replication-dependent chromosomal fragmentation are being tested and more are suspected.
Exogenous DNA damage and chromosomal lesions: carcinogenesis and anti-cancer treatment
Strong mutagens induce cancer because they cause genome instability (point mutations) directly. However, many powerful carcinogens are DNA-damaging agents, rather than mutagens, with the mechanisms of carcinogenesis completely unclear. For example, UV light is a potent carcinogen, but the only DNA lesions it directly induces, called pyrimidine dimers, are not mutagenic and are completely removable by the nucleotide-excision repair. The expectation, therefore, is that UV light would inhibit cell growth (until all the pyrimidine dimers are repaired), but would have no consequence on cells' viability or genome stability. Yet, UV light in sufficiently high doses eventually kills the cells or causes gross chromosomal rearrangements in them, which leads to cancerous transformation.
The UV-induced DNA lesions that either kill the cells or cause gross chromosomal rearrangements must be dramatically different from the original pyrimidine dimers. We study the nature of these aggravated DNA lesions, which we call "chromosomal lesions". We already know that the formation of DNA damage-induced chromosomal lesions is intimately linked to replication fork encounters with primary DNA lesions (pyrimidine dimers), either unrepaired, or in the process of repair. The multiple pathways of replication fork stalling at DNA lesions, replication fork reversal and restart, as well as disintegration, is the focus of our research. Our findings have direct relevance for the mechanisms of both carcinogenesis and the action of the most potent anti-cancer drugs.
Replication forks as the focal points of genome instability and cytocidal treatments
Our studies highlight replication forks as the focal points of the cytotoxic or genome-destabilizing action of DNA damaging treatments. However, DNA damage does not have to be direct to kill the cells or to transform them. One of the most efficient and yet enigmatic ways to kill the cells of any type is to manipulate the pools of DNA precursors. The phenomenon of thymineless death, when the cells cannot synthesize the DNA precursor dTTP, has been known for over five decades now and lies in the basis of several anti-cancer and anti-microbial treatments, yet its chromosomal mechanisms are completely unclear. Recently we and others have found that the replication origin DNA is degraded in E. coli undergoing thymineless death, this degradation being dependent, surprisingly, on the ability of the cells to restart stalled replication forks by recombinational repair. Thus, the puzzle of thymineless death, instead of being resolved, became even more bizarre! The solution of the thymineless death mystery will have a direct relevance for both the mechanisms of genomic instability and the efficacy of anti-cancer treatments.
Hydroxyurea offers a general way of inhibiting DNA synthesis in any type of cells, by blocking ribonucleotide reductase, the enzyme responsible for production of all four DNA precursors. Hydroxyurea is an effective treatment against myeloproliferative disorders and sickle-cell anemia. Hydroxyurea was always assumed to be cytostatic (inhibiting, but not killing the cells), but recently we and others have found that it can be extremely cytotoxic. The mechanisms and conditions of hydroxyurea cytotoxicity may be linked to its efficacy as a drug and will be studied further. Again, our experimentation in this tractable modal system will inform and help our efforts to treat life-threatening health conditions.
Figure 1. The color screen for synthetic lethal combinations as a tool to identify mutants with interesting properties.
Figure 2. Pulsed-field gel electrophoresis as a method to quantify chromosomal fragmentation.
Fig. 3. Genome marker-frequency profiles reveals disappearance of the origin region in the chromosome of cells undergoing thyminless death (yellow), compared to control cells (dark blue or pink). The profile of non-replicating cells is in gray. (click for an enlarged image)
Amado L. and Kuzminov A. (2013) Low-molecular-weight DNA replication intermediates in Escherichia coli: mechanism of formation and strand specificity. J. Mol. Biol., http://dx.doi.org/10.1016/j.jmb.2013.07.021
Khan S.R. and Kuzminov A. (2013) Trapping and breaking of in vivo nicked DNA during pulsed-field gel electrophoresis. Anal. Biochem. Jun 13. [Epub ahead of print]
Kuzminov A. (2013) Inhibition of DNA synthesis facilitates expansion of low-complexity repeats: is strand slippage stimulated by transient local depletion of specific dNTPs? BioEssays 35: 306-313.
Rotman E., Kouzminova E., Plunkett III G. and Kuzminov A. (2012) Genome of enterobacteriophage Lula/phi80 and insights into its ability to spread in the laboratory environment. J. Bacteriol., 194: 6802-6817.
Kuong K.J and Kuzminov A. (2012) Disintegration of nascent replication bubbles during thymine starvation triggers RecA- and RecBCD-dependent replication origin destruction. J. Biol. Chem., 287: 23958-23970.
Kouzminova E.A. and Kuzminov A. (2012) Chromosome demise in the wake of ligase-deficient replication. Mol. Microbiol., 84: 1079-1096. Cover story.
Khan S.R. and Kuzminov A. (2012) Replication forks stalled at UV-lesions are rescued via RecA- and RuvABC-catalyzed disintegration in Escherichia coli. J. Biol. Chem., 287: 6250–6265.
Kuzminov A. (2011) Chapter 7.2.6, Homologous Recombination—Experimental Systems, Analysis, and Significance. In A. Böck, R. Curtiss III, J. B. Kaper, P. D. Karp, F. C. Neidhardt, J. M. Slauch, and C. L. Squires (ed.), EcoSal—Escherichia coli and Salmonella: Cellular and Molecular Biology. ASM Press, Washington, DC. http://www.ecosal.org. doi: 10.1128/ecosal.7.2.6
Rotman E., Amado L. and Kuzminov A. (2010) Unauthorized Horizontal Spread in the Laboratory Environment: The Tactics of Lula, a Temperate Lambdoid Bacteriophage of Escherichia coli. PLoS ONE, 5: 1-13 (e11106).
Kuong K.J. and Kuzminov A. (2010) Stalled replication fork repair and misrepair during thymineless death in Escherichia coli. Genes-to-Cells, 15: 619–634. PMID: 20465561
Budke B. and Kuzminov A. (2010) Production of clastogenic DNA precursors by the nucleotide metabolism in Escherichia coli. Mol. Microbiol., 75: 230-245. PMID: 19943897
Amado L. and Kuzminov A. (2009) Polyphosphate Accumulation in Escherichia coli in response to defects in DNA metabolism. J Bacteriol. 191: 7410-7416. PMID: 19837803
Kuong K.J. and Kuzminov A. (2009) Cyanide, Peroxide and Nitric Oxide Formation in Solutions of Hydroxyurea Causes Cellular Toxicity and May Contribute to Its Therapeutic Potency. J. Mol. Biol. 390: 845-862. PMID: 19467244
Rotman E., Bratcher P. and Kuzminov A. (2009) Reduced LPS phosphorylation in Escherichia coli lowers the elevated ori/ter ratio in seqA mutants. Mol. Microbiol. 72: 1273-1292. PMID: 19432803
Ting H., Kouzminova E.A. and Kuzminov A. (2008) Synthetic lethality with the dut defect in Escherichia coli reveals layers of DNA damage of increasing complexity due to uracil incorporation. J. Bacteriol. 190: 5841-5854. PMID: 18586941
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. Mol. Microbiol., 68: 202-215. PMID: 18312272
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: 6976-6988. PMID: 17616589
Budke B. and Kuzminov A. (2006) Hypoxanthine incorporation is non-mutagenic in Escherichia coli. J. Bacteriol., 188: 6553-6560. PMID: 16952947
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: 22635-22646. PMID: 16772291
Lukas L. and Kuzminov A. (2006) Chromosomal fragmentation is the major consequence of the rdgB defect in Escherichia coli. Genetics, 172: 1359-1362. PMID: 16322510
Kouzminova E.A. and Kuzminov A. (2006) Fragmentation of replicating chromosomes triggered by uracil in DNA. J. Mol. Biol. 355: 20-33. PMID: 16297932
Shi I.Y., Stansbury J. and Kuzminov A. (2005) Defect in Acetyl-CoA<—>Acetate Pathway poisons recombinational repair-deficient mutants of Escherichia coli. J. Bacteriol. 187: 1266-1275. PMID: 15687190
Kuzminov A. and Stahl F.W. (2005) Chapter 19. Overview of Homologous Recombination and Repair Machines. In: The Bacterial Chromosome. ed: Higgins N.P., Washington, D.C.: ASM Press, 349-367.
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: 16262-16267. PMID: 15531636
Kouzminova E.A. and Kuzminov A. (2004) Chromosomal fragmentation in dUTPase-deficient mutants of Escherichia coli and its recombinational repair. Mol. Microbiol. 51(5): 1279-1295. PMID: 14982624
Bradshaw J.S. and Kuzminov A. (2003) RdgB acts to avoid chromosome fragmentation in Escherichia coli. Mol. Microbiol. 48(6): 1711-1725. PMID: 12791149
Miranda A. and Kuzminov A. (2003) Chromosomal lesion suppression and removal in Escherichia coli via linear DNA degradation. Genetics 163: 1255-1271. PMID: 12702673
Kuzminov A. (2001) DNA replication meets genetic exchange: Chromosomal damage and its repair by homologous recombination. Proc. Natl. Acad. Sci. USA 98(15): 8461-8468. PMID: 11459990
Kuzminov A. (2001) Single-strand interruptions in replicating chromosomes cause double-strand breaks. Proc. Natl. Acad. Sci. USA 98(15): 8241-8246. PMID: 11459959