Malkova Lab
Research Summary
Repair of double-strand breaks (DSB): Break-Induced Replication (BIR)
The work in our lab is aimed on investigation of the mechanisms of double-strand break (DSB) repair. DNA double-strand breaks (DSBs) pose a formidable challenge to genome integrity and must therefore be removed promptly. While some DSB repair pathways are precise, others, most notably break-induced replication (BIR), can lead to potentially deleterious genome alterations associated with cancer.
While BIR is the primary pathway for repairing one-ended DSBs arising from collapsed replication forks and eroded telomeres, it frequently leads to loss of heterozygosity (LOH), genetic mutations, and gross chromosomal rearrangements, all of which are hallmarks of cancer. In particular, BIR generates clusters of mutations (“kataegis”) that can drive the neoplastic transformation of cells. BIR plays a significant role at the onset of carcinogenesis, when cells undergo a massive collapse of replication forks, and which are repaired by BIR. Importantly, BIR drives the alternative lengthening of telomere (ALT) mechanism and work by others provide strong support for the overarching hypothesis that bursts of genetic instability associated with BIR helps to drive the evolution of the cancer genome and can lead to adverse outcomes including drug resistance and metastasis.
What is BIR?
BIR mechanism and its genome destabilizing outcomes. BIR (left) leads to genetic instabilities (right) including loss of heterozygosity, mutation clusters (kataegis; asterisks denote mutations), and chromosomal rearrangements. BIR also mediates alternative lengthening of telomeres in ~15% of human tumors. BIR:
Is up to 1,000-fold more mutation-prone compared to chromosomal replication
Leads to loss of heterozygosity (LOH)
Creates a substrate for kataegis
Leads to complex chromosomal rearrangements
Can drive alternative lengthening of telomeres (ALT)
In the lab, we are interested in how imprecise or faulty repair of DSBs leads to structural genomic variations including mutations, copy number variations (CNVs) and chromosomal rearrangements similar to those that cause genetic diseases and cancer in humans. We are using sensitive genetic assays, direct physical methods and genomic approach to unravel the mechanisms of genetic instabilities resulting from BIR, and the effects of various environmental factors in the development of such instabilities. Notably, by using genetics, cell and molecular biology, and biochemical reconstitution, we have shown that BIR is initiated by the invasion of a DNA end into a homologous template followed by extensive DNA synthesis that proceeds via a migrating “DNA bubble”.
However, many details of the mechanism of BIR responsible for its destabilizing effects, as well as its role in promoting genetic instabilities leading to cancer remain unclear. We aim to fill these gaps in our knowledge by investigating BIR in yeast and eventually in humans. In addition, we use our knowledge obtained in yeast to analyze human cancer genome databases to determine the role of BIR in promoting genomic instabilities leading to cancer.
Selected Publications
Complete list of publications available in Google Scholar
Musmaker, K., Wells, J., Tsai, M. C., Comeron, J. M., & Malkova, A. (2024). Alternative Lengthening of Telomeres in Yeast: Old Questions and New Approaches. Biomolecules, 14(1), 113. https://doi.org/10.3390/biom14010113. PMCID: PMC10813009
Lee, R. S., Geronimo, C. L., Liu, L., Twarowski, J. M., Malkova, A., & Zakian, V. A. (2023). Identification of the nuclear localization signal in the Saccharomyces cerevisiae Pif1 DNA helicase. PLoS genetics, 19(7), e1010853. https://doi.org/10.1371/journal.pgen.1010853 . PMCID: PMC10399864.
Osia B., Twarowski J., Jackson T., Lobachev K., Liu L., Malkova A. (2022). Migrating bubble synthesis promotes mutagenesis through lesions in its template. Nucleic Acids Res., 50: 6870-6889. https://doi.org/10.1093/nar/gkac520. PMCID: PMC9262586.
We discovered a new source of mutagenic single-strand DNA within the migrating D-loop of BIR.
Kockler Z., Comeron J., Malkova A. (2021). A unified alternative telomere-lengthening pathway in yeast survivor cells. Molecular Cell, 81:1816-1829. https://doi.org/10.1016/j.molcel.2021.02.004. PMCID: PMC8052312.
By utilizing a combination of computer modeling, population genetics, and ultra-long sequencing, we have uncovered, for the first time, 'molecular milestones' representing different steps in the establishment of ALT in yeast.
Liu L., Yan Z., Osia B., Twarowski J., Sun L., Kramara J., Lee R., Kumar S., Elango R., Li H., Dang W., Ira G., Malkova A. (2021). Tracking break-induced replication shows that it stalls at roadblocks. Nature, 590: 655-659. https://doi.org/10.1038/s41586-020-03172-w. PMCID: PMC8219245.
Using our novel droplet-digital PCR-based AMBER assay, we identified critical BIR intermediates, characterized the impact of roadblocks on BIR, and measured BIR kinetics at unprecedented resolution.
Osia B., Alsulaiman T., Jackson T., Kramara J., Oliveira S., Malkova A. (2021). Cancer cells are highly susceptible to accumulation of templated insertions linked to MMBIR. Nucleic Acids Res., 49: 8714-8731. https://doi.org/10.1093/nar/gkab685. PMCID: PMC8421209.
This is the first demonstration that microhomology-mediated BIR (MMBIR) events are frequent in cancers but rare in non-cancerous cells.
Elango R., Osia B., Malc E., Mieczkowski P., Roberts S., Malkova A. (2019). Repair of base damage within break-induced replication intermediates promotes kataegis associated with chromosome rearrangements. Nucleic Acids Res, 47:9666-9684. https://doi.org/10.1093/nar/gkz651. PMCID: PMC6765108.
This is the first demonstration that in the presence of human APOBEC 3A, BIR leads to the formation of mutation clusters similar to “kataegis” observed in cancer.
Elango R., Zhang S., Jackson J., DeCata J., Ibrahim Y., Pham N., Liang D., Sakofsky C., Lobachev K., Ira G., Malkova A. (2017). Break-induced replication promotes formation of lethal joint molecules dissolved by Srs2. Nature Communications, 8:1790 -1805. https://doi.org/10.1038/s41467-017-01987-2. PMCID:PMC5702615.
This is the first demonstration that BIR promotes the formation of lethal intermediates. resulting from the strand invasion of long single-strand DNA accumulated during BIR into the homologous chromosome.
Sakofsky C., Ayyar S., Deem A. K., Chung W. H., Ira G., Malkova A. (2015). Translesion Polymerases Drive Microhomology-Mediated Break-Induced Replication Leading to Complex Chromosomal Rearrangements. Molecular Cell, 60: 860-872. https://doi.org/10.1016/j.molcel.2015.10.041. PMCID: PMC26669261.
This is the first demonstration that interruption of BIR promotes switch to microhomology-mediated BIR (MMBIR), involving template switching at positions of microhomology.
Sakofsky C. J., Roberts S. A., Malc E., Mieczkowski P. A., Resnick M. A., Gordenin D. A., Malkova A. (2014). Break-induced replication is a source of mutation clusters underlying kataegis. Cell Reports, 7: 1640-1648. https://doi.org/10.1016/j.celrep.2014.04.053. PMCID: PMC24882007.
This is the first demonstration that in the presence of alkylating damage BIR leads to the formation of mutation clusters similar to those observed in cancer.
Vasan S., Deem A., Ramakrishnan S., Argueso J. L., Malkova A. (2014). Cascades of genetic instability resulting from compromised break-induced replication. PLoS Genetics, 10, e1004119. https://doi.org/10.1371/journal.pgen.1004119. PMCID: PMC3937135.
This is the first demonstration that interruption of BIR leads to cascades of genome rearrangements similar to cycles of non-reciprocal translocations detected in cancer, specifically, to cycles of non-reciprocal translocations.
Saini N., Ramakrishnan S., Ayyar S., Zhang Y., Deem A., Elango R., Ira G., Harber J., Lobachev K., Malkova A. (2013). Migrating bubble during break-induced replication drives conservative DNA synthesis. Nature, 502: 389-392. https://doi.org/10.1038/nature12584. PMCID: PMC3804423.
This is the first demonstration that BIR proceeds by a bubble-migrating DNA synthesis resulting in the conservative inheritance of newly synthesized DNA.
Wilson M. A., Kwon Y., Xu Y., Chung W. H., Chi P., Niu H., Mayle R., Chen X., Malkova A., Sung P., Ira G. (2013). Pif1 helicase and Polδ promote recombination-coupled DNA synthesis via bubble migration. Nature, 502: 393-396. https://doi.org/10.1038/nature12585. PMCID: PMC24025768.
This is the first demonstration that BIR proceeding by a migration bubble is carried by DNA Polymerase δ and Pif1 helicase.
Deem A., Keszthelyi A., Blackgrove T., Vayl A., Coffey B., Mathur R., Chabes A., Malkova A. (2011). Break-induced Replication is highly inaccurate. PLoS Biology, 9(2), e1000594. https://doi.org/ARTN e1000594. PMCID: PMC3039667.
This is the first demonstration that BIR is ~1000 times more mutagenic than normal DNA replication.
VanHulle K., Lemoine F., Narayanan V., Downing B., Hull K., McCullough C., Bellinger M., Lobachev K., Malkova A. (2007). Inverted DNA repeats channel repair of distant double-strand breaks into chromatid fusions and chromosomal rearrangements. Mol. Cell. Biol, 27: 2601-2614. https://doi.org/10.1128/MCB.01740-06. PMCID: PMC 17242181.
This is the first demonstration that in the absence of Rad51, BIR always leads to chromosome rearrangements.
Malkova, A., Ivanov, E., Haber, J. (1996). Double-strand break repair in the absence of RAD51 in yeast: a possible role for break-induced DNA replication. Proc Natl Acad Sci, 93: 7131-7136. https://doi.org/10.1073/pnas.93.14.7131. PMCID: PMC38948.
Here, we describe (for the first time) break-induced replication (BIR) in eukaryotes, specifically Rad51-independent BIR.