| NSF Org: |
MCB Division of Molecular and Cellular Biosciences |
| Recipient: |
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| Initial Amendment Date: | August 12, 2016 |
| Latest Amendment Date: | September 11, 2017 |
| Award Number: | 1615335 |
| Award Instrument: | Continuing Grant |
| Program Manager: |
Candi Phoebe Lostroh
MCB Division of Molecular and Cellular Biosciences BIO Directorate for Biological Sciences |
| Start Date: | August 15, 2016 |
| End Date: | July 31, 2020 (Estimated) |
| Total Intended Award Amount: | $690,000.00 |
| Total Awarded Amount to Date: | $690,000.00 |
| Funds Obligated to Date: |
FY 2017 = $459,999.00 |
| History of Investigator: |
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| Recipient Sponsored Research Office: |
926 DALNEY ST NW ATLANTA GA US 30318-6395 (404)894-4819 |
| Sponsor Congressional District: |
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| Primary Place of Performance: |
505 10th Street Atlanta GA US 30332-0415 |
| Primary Place of
Performance Congressional District: |
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| Unique Entity Identifier (UEI): |
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| Parent UEI: |
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| NSF Program(s): | Genetic Mechanisms |
| Primary Program Source: |
01001718DB NSF RESEARCH & RELATED ACTIVIT |
| Program Reference Code(s): |
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| Program Element Code(s): |
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| Award Agency Code: | 4900 |
| Fund Agency Code: | 4900 |
| Assistance Listing Number(s): | 47.074 |
ABSTRACT
A break in both strands of the DNA double helix is one of the most harmful DNA lesions because if not properly repaired it leads to mutations, chromosome rearrangements or cell death. The safest way to repair a DNA double-strand break (DSB) is through a process called homologous recombination (HR). The known mechanisms of HR utilize an intact copy of the DNA sequence as a template to retrieve the information lost at the site of a DSB. However, recent results, which will be expanded in this study, indicate a new role of RNA in this process of DNA repair. Therefore, the study will provide important new biological insights to better understanding the physiological function of RNA in DNA repair, and the impact of RNA on genome maintenance and evolution. A postdoctoral fellow, a graduate student and several undergraduate students will be involved in the research. The work and findings of this proposal will be integrated in class topics and activities for many graduate and undergraduate students. Through the inclusion of a Research Experience for Teachers (RET), the project will also target students from a local, 100%-minority High School to support student interest and participation in Science, Technology, Engineering and Math (STEM) programs.
To characterize the mechanism of RNA-DNA recombination, novel genetic systems will be developed to study repair of chromosomal double-strand breaks (DSBs) by homologous transcript RNA in budding yeast cells. One Objective of this project will be to identify the DNA polymerization function/s that use RNA as template in RNA-DNA recombination. Another focus will be to examine whether the process of DSB repair by transcript RNA can be visualized in cells arrested at two distinct points in the cell cycle: before and after DNA replication. It was shown that DSB repair by transcript RNA in yeast cells is initiated efficiently, but it is quickly suppressed by cellular ribonuclease H enzymes. Here, refined genetic systems to detect DSB repair by RNA will be utilized to determine the efficiency of DSB repair by transcript RNA in wild-type and ribonuclease-defective cells. Overall, the work of this project will help to better understand the conditions and mechanism in which transcript RNA is a preferred direct template for DSB repair in cells.
This project is funded by the Genetic Mechanisms Program in the Division of Molecular and Cellular Biosciences.
PUBLICATIONS PRODUCED AS A RESULT OF THIS RESEARCH
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PROJECT OUTCOMES REPORT
Disclaimer
This Project Outcomes Report for the General Public is displayed verbatim as submitted by the Principal Investigator (PI) for this award. Any opinions, findings, and conclusions or recommendations expressed in this Report are those of the PI and do not necessarily reflect the views of the National Science Foundation; NSF has not approved or endorsed its content.
A few years ago, we discovered that RNA transcripts generated inside cells serve as template for DNA double-strand break (DSB) repair, directly, as well as indirectly via a cDNA intermediate, transferring genetic information to chromosomal DNA in budding yeast (PMID: 25186730). The work of this NSF project supported our findings that transcript RNA is used as a template for repair of DNA breaks in an inverse strand-exchange mechanism mediated by the recombination protein Rad52 (PMID: 28602639). Via a series of molecular, genetic, and biochemical assays we defined and characterized the genetic controls of three distinct mechanisms how RNA can recombine with DNA. These are: RNA-templated DSB repair, cDNA-templated DSB repair, and RNA-templated DNA modification, discovered in this project, which directly modifies DNA with transcript RNA in the absence of an induced DSB. We found that only RNA-templated DNA modification is Rad52 independent, providing the first proof of a homologous-recombination process in yeast without the need for recombination proteins. We demonstrate that constitutive transcription preceding DSB induction significantly enhances the frequency of RNA-templated DSB repair. We discovered a major role of the translesion DNA polymerase ζ in RNA-templated DSB repair, and an essential role of Pol ζ in RNA-templated DNA modification. Differently, we found that the reverse-transcriptase activity of the yeast retrotransposon is only required for cDNA-templated DSB repair. Because Pol ζ is error prone, RNA-templated DSB repair and RNA-templated DNA modification may be a mutagenic process suggesting that single nucleotide polymorphisms may transfer from RNA to DNA. Interestingly, our results show that mismatch repair with MSH2/MSH6 is important for RNA-templated DSB repair, possibly to repair mismatches generated by Pol ζ. Furthermore, we found that cDNA-templated DSB repair is dependent on clippase functions (RAD1/10-MSH2/3), while this is not the case for RNA-templated DSB repair and RNA-templated DNA modification. Our findings illustrate a powerful role of RNA in directly (RNA-templated DSB repair and RNA-templated DNA modification) and indirectly (cDNA-templated DSB repair) templating genomic modifications as a driver of genome stability/instability.
The work of this project played fundamental role for teaching, training, and mentoring of many students at all levels, postdoctoral fellows, a research scientist, and a high school teacher of biological sciences with numerous high school students. The results of the project have been broadly disseminated to the scientific community and the society mainly via numerous publications and a large number of presentations at national and international conferences and as seminar talks at national and international institutions.
Last Modified: 11/18/2020
Modified by: Francesca Storici
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