Role of R-loops in DNA double-strand break repair and cancer
1 PhD project offered in the IPP winter call Molecular Mechanisms in Genome Stability & Gene Regulation
Scientific Background
The integrity of DNA is essential for normal cellular functions. The correct base-pairing between the opposite strands of the DNA double helix allows double-stranded DNA (dsDNA) to maintain a stable B-form conformation. However, cells unpair and temporarily open dsDNA to carry out processes like replication and transcription. Inefficient execution and/or termination of these events can result in the generation of non-B form structures such as hairpins, cruciforms, G-quadruplex structures, R-loops, etc. R-loops are three-stranded DNA
hybrid structures where RNA is annealed to one of the complementary DNA strands in a DNA duplex, displacing the other non-complementary DNA strand. While R-loops are known for their detrimental effect on gene expression and genome maintenance, growing evidence suggests a regulatory role of R-loops in gene regulation, promoting DNA repair, maintaining telomere length, and immunoglobulin class switch recombination. Persistent R-loops are impediments to replication fork progression and, if not removed in a timely manner, can cause DNA double-strand breaks (DSBs) due to transcription-replication collisions.
Telomeres are long repetitive DNA sequences (TTAGGG) that are present at the ends of chromosomes. Telomeres shorten with each cell division due to the end-replication problem, which limits the number of times a cell can divide before it ceases to proliferate. To achieve unrestricted cellular proliferation, cancer cells in 10-15% of all cancers utilize a recombination-mediated process known as alternative lengthening of telomeres (ALT) to maintain a critical length of telomeres and avoid cell death. During ALT, shortened telomeres are resected to produce single-stranded DNA (ssDNA), which invades other telomeric duplexes to form a displacement loop or D-loop. The D-loops structurally resemble R-loops, except all three strands are DNA strands. In a subsequent step, the invaded strand is extended by DNA synthesis, using another telomeric region as a template to extend the shortened telomeres. Since telomeric ends resemble one-sided DSBs, ALT utilizes a mechanism similar to homologous recombination (HR), a predominant DSB repair pathway, to extend the telomeres.
PhD Project: Investigating the life cycle of RAD51AP1-mediated R-loops
Telomeres, initially thought to be transcriptionally inactive, are transcribed and produce non-coding RNA known as TERRA (telomeric repeat-containing RNA). TERRA RNA can hybridize with telomeric DNA to form telomeric R-loops (tR-loops). These tR-loops are elevated in ALT-positive cancers and have been directly implicated in the regulation of ALT. Specifically, optimal levels of tR-loops are required to induce constitutive replication stress for recombination to occur between telomeres via ALT. Too many or too few tR-loops are detrimental to the survival of ALT cancer cells.
RAD51-associated protein 1 (RAD51AP1) is a pro-HR factor that functions with RAD51 recombinase to promote D-loop formation during HR. Cells lacking RAD51AP1 exhibit hypersensitivity to DSB-causing genotoxic agents, and RAD51AP1 is found to be upregulated in various cancers. In recent years, RAD51AP1 has emerged as a necessary ALT factor, primarily due to its capacity to form R-loops via its RNA-strand invasion activity into dsDNA. RAD51AP1 contributes to ALT at two levels. First, RAD51AP1 forms telomeric R-loops using TERRA to maintain a critical level of replication stress to sustain an optimal level of ALT. Second, RAD51AP1-mediated R-loops aid in proficient D-loop formation by transiently opening the dsDNA duplex to facilitate efficient DNA strand invasion. Similarly, during HR, when DSBs occur in actively transcribed genes, the RNA transcripts are used by RAD51AP1 to make R-loops in the donor duplex to promote efficient D-loop formation. The transitory DNA structure, where both R- and D-loops exist in the same duplex, is known as DR-loops. In light of these recent findings, we have only begun to appreciate the multifaceted roles of RAD51AP1 in DSB repair and ALT. Many outstanding questions regarding RAD51AP1’s function in DSB repair and ALT remain unanswered.
In this project, we will primarily investigate RAD51AP1’s R-loop formation activity using in vitro biochemistry and determine the mechanistic basis of RAD51AP1 functions in DSB repair and ALT. Specifically, we will elucidate the mechanisms of genomic and telomeric R-loop formation by RAD51AP1, the transition of R-loops to D-loops (DR-loop switch), and DNA synthesis post-DR-loop switch. We will tackle the following questions to discover RAD51AP1’s function in maintaining genomic stability and ALT.
How does RAD51AP1 form genomic and telomeric R-loops?
What is the mechanism of the R-to-D loop switch?
How does DNA synthesis initiate from DR-loops?
To achieve these aims, we will use bulk biochemistry and single-molecule imaging methods. We will form R-loops (both genomic and tR-loops) outside the cells using synthetic DNA and purified protein(s). To gain unprecedented details of R-loop formation by RAD51AP1, we will study these reactions at the single-molecule level using optical tweezers with confocal microscopy (C-trap, Lumicks). In summary, this project will uncover the molecular mechanisms of the life cycle of R-loops to deepen our understanding of DSB repair and ALT, illuminating key aspects of cancer development and survival.
We are looking for motivated PhD student with strong interest in the mechanism of DNA damage response, genomic instability, and cancer development. Ideally, the interested candidate will have some prior experience of molecular biology for cloning, protein expression and purification, and biochemical assays. However, lack of experience in these techniques should not stop you from applying if you are strongly interested in learning the requisite skills. You bring your enthusiasm and determination; we will do the rest.
This project will be part of the RTG on R-loop Regulation in Robustness and Resilience (4R).
If you are interested in this project, please select Anand as your group preference in the IPP application platform.
Publications relevant to this project
Petermann E, Lan L & Zou L (2022) Sources, resolution and physiological relevance of R-loops and RNA–DNA hybrids. Nat. Rev. Mol. Cell Biol. 23, 521–540 Link
Ouyang J, Yadav T, Zhang JM, Yang H, Rheinbay E, Guo H, Haber DA, Lan L, Zou L (2021) RNA transcripts stimulate homologous recombination by forming DR-loops. Nature594, 283–288 Link
Yadav T, Zhang JM, Ouyang J, Leung Q, Simoneu A, Zou L(2022) TERRA and RAD51AP1 promote alternative lengthening of telomeres through an R- to D-loop switch. Mol. Cell82, 3985-4000.e4 Link
Kaminski N, Wondisford AR, Kwon Y, Lynskey ML, Bhargava R, Barroso-Gonzáles J, García-Expósito L, He B, Xu M, Mellacheruvu D, Watkins SC, Modesti M, Miller KM, Nesvizhskii AI, Zhang H, Sung P, O'Sullivan RJ (2022) RAD51AP1 regulates ALT-HDR through chromatin-directed homeostasis of TERRA. Mol. Cell82, 4001-4017.e7. Link