OB-Folds and Genome Maintenance: Targeting Protein–DNA Interactions for Cancer Therapy

SMC complexes play key roles in genome maintenance, where they ensure efficient genome replication and segregation. The SMC complex Smc5/6 is a crucial player in DNA replication and repair, yet many molecular features that determine its roles are unclear. Here, we use single-molecule microscopy to investigate Smc5/6's interaction with DNA. We find that Smc5/6 forms oligomers that dynamically redistribute on dsDNA by 1D diffusion and statically bind to ssDNA. Using combined force manipulation and single-molecule microscopy, we generate ssDNA-dsDNA junctions that mimic structures present in DNA repair intermediates or replication forks. We show that Smc5/6 accumulates at these junction sites, stabilizes the fork, and promotes the retention of RPA. Our observations provide a model for the complex's enrichment at sites of replication stress and DNA lesions from where it coordinates the recruitment and activation of downstream repair proteins.

Single stranded DNA (ssDNA) binding proteins (SSBPs), are known key players of DNA damage response (DDR) pathway and play an essential role in stabilising fragile ssDNA generated during DNA replication, transcription and repair. The canonical SSBP is the heterotrimeric Replication Protein A (RPA) which is involved in a number of key cellular processes including replication and repair via Homologous Recombination (HR) in the course of DNA damage. Our lab recently described two new SSBPs, termed SSB1 and SSB2 (also known as NABP2/OBFC2B/SOSS-B1 and NABP1/OBFC2A/SOSSB-2, respectively) which form independent co-complexes with two additional proteins, the Integrator complex subunit 3 (INTS3) and the chromosome 9 open reading frame 80 (C9ORF80), a small acidic 104 residue polypeptide. Previously, we demonstrated that whilst Ssb1/Nabp2 KO in mouse caused perinatal lethality, Ssb2/Nabp1 KO did not lead to any phenotypic abnormalities. Interestingly, ablation of Ssb1 led to stabilisation of Ssb2 and vice-versa, indicating functional redundancy between these two proteins. This was recently demonstrated in-vivo by the generation of Ssb1 and Ssb2 (together referred as Ssb1/2) double-knockout (DKO) mice, which caused early embryonic lethality in a constitutive model and acute bone marrow failure and intestinal atrophy using the inducible Rosa26-CreERT2 system. To delineate the functional redundancy between these two proteins at the molecular level, we have generated inducible DKO mouse embryonic fibroblasts (MEFs) using the Rosa26-CreERT2 system, which will be described in the first research chapter. We found that cumulative loss of Ssb1/2 in the primary as well as SV40-immortalised MEFs led to acute proliferation arrest and cell death following TAM administration. This was associated with accumulation of genomic instability via endogenous replication stress. Although loss of Ssb1/2 in-vivo and in-vitro is associated with accumulation of R-loops, the overall DKO phenotype was not able to be rescued with overexpression of RNaseH1, which resolves R-loops. Additionally, we investigated the roles of Ssb1/2 following treatment with different DNA damaging agents to determine their roles in the DDR system. Interestingly, DDR signalling in DKO was normal following ionizing radiation, ultraviolet C and camptothecin but with hydroxyurea treatment that causes replication stress, we observed a delayed signalling response in DKO. Together, this chapter defines the phenotypic changes that take place in-vitro when Ssb1 and Ssb2 are deleted. The second research chapter describes our finding that loss of Ssb1 and Ssb2 together leads to reduced levels of several Integrator components and thus, has an equivalent profound effect on the misprocessing of the Sm- associated small nuclear RNAs (snRNAs) to that of the Integrator catalytic components- IntS9 and IntS11. Here, we show that upregulated snRNAs are not only misprocessed, but extend up to several hundred to a thousand base pairs past their native termination site, and are polyadenylated. Additionally, we demonstrate that loss of Ssb1/2 led to changes in the dynamics of alternative splicing, likely due to perturbation of the splicing machinery by aberrant snRNAs. We further show that a number of regulators of transcription and the cell cycle are affected by these changes, which might contribute to the loss of viability observed in DKO cells. Together, these findings reveal the critical role of Ssb1/2 and their association with the Integrator complex in regulating cellular proliferation and spliceosomal function. The third chapter of this thesis further investigates the intestinal atrophy observed upon loss of Ssb1 and Ssb2 in the DKO mice from our lab. For this, we have generated a small intestine (SI) specific Ssb1/2 DKO mouse- the VillinCreERT2 Ssb1flox/flox; Ssb2flox/flox model. This mouse model is a unique system to study the undefined roles of Ssb1/2 in the small intestine (SI) by bypassing the confounding effects of the bone marrow phenotype in the ubiquitous Rosa26-CreERT2 DKO model. We have found that loss of intestinal Ssb1/2 leads to exhaustion of the stem cells in the crypts, resulting in loss of the normal crypt-villus axis anatomy which causes acute morbidity within six days of induction. Interestingly, the stem cells are pushed to proliferate immediately after the loss of Ssb1 and Ssb2, followed by the exhaustion of these cells. This is demonstrated by sequential proliferation studies using the known thymidine analogue 5-bromo-2’deoxyuridine (BrdU) as well as quantitative reverse transcription polymerase chain reaction (qRT-PCR). Therefore through this model, we have demonstrated a fundamental role of Ssb1/2 in the maintenance of intestinal homeostasis. In conclusion, through the inducible abrogation of two SSBPs- Ssb1 and Ssb2 together, we have demonstrated several novel roles of these proteins in the maintenance of genomic stability both in-vitro and in-vivo that were previously masked in single KO studies. Further, we have defined the molecular mechanisms underlying the acute lethality observed upon abrogation of these two proteins.

Chromatin-based DNA damage response (DDR) pathways are fundamental for preventing genome and epigenome instability, which are prevalent in cancer. Histone acetyltransferases (HATs) and histone deacetylases (HDACs) catalyze the addition and removal of acetyl groups on lysine residues, a post-translational modification important for the DDR. Acetylation can alter chromatin structure as well as function by providing binding signals for reader proteins containing acetyl-lysine recognition domains, including the bromodomain (BRD). Acetylation dynamics occur upon DNA damage in part to regulate chromatin and BRD protein interactions that mediate key DDR activities. In cancer, DDR and acetylation pathways are often mutated or abnormally expressed. DNA damaging agents and drugs targeting epigenetic regulators, including HATs, HDACs, and BRD proteins, are used or are being developed to treat cancer. Here, we discuss how histone acetylation pathways, with a focus on acetylation reader proteins, promote genome stability and the DDR. We analyze how acetylation signaling impacts the DDR in the context of cancer and its treatments. Understanding the relationship between epigenetic regulators, the DDR, and chromatin is integral for obtaining a mechanistic understanding of genome and epigenome maintenance pathways, information that can be leveraged for targeting acetylation signaling, and/or the DDR to treat diseases, including cancer.

DNA double strand breaks (DSBs) result in activation of several key DNA damage response (DDR) kinases including ATM, ATR, and DNA-PK. These protein kinases not only promote DNA damage-induced checkpoint control, but also facilitate DSB repair in humans. Thus, these DDR kinases have become promising drug targets for cancer therapy. However, the benefits of targeting DDR kinases remain to be realized, in part due to the lack of predictive biomarkers. By undertaking CRISPR screens with inhibitors targeting key DDR kinases, we obtained a global and unbiased view of genetic interactions with DDR inhibition. Additionally, we compared the synergistic effects of combining different DDR inhibitors and found that ATM and PARP inhibitors showed remarkable synergy, which depends on the dominant negative function of ATM inhibition. Moreover, to provide comprehensive and unbiased perspective of DDR signaling pathways, we performed 30 fluorescence-activated cell sorting–based genome-wide CRISPR screens with antibodies recognizing distinct endogenous DNA damage–signaling proteins to identify new regulators involved in DNA damage response (DDR). We discovered that proteasome-mediated processing is an early and prerequisite event for cells to trigger camptothecin- and etoposide-induced DDR signaling. Furthermore, we identified PRMT1 and PRMT5 as new modulators that regulate ATM protein level. Additionally, we discovered that GNB1L is a master regulator of DDR signaling via its role as a co-chaperone for PIKK proteins. Collectively, these screens offer a rich resource for further investigation of DDR, which may provide insight into strategies of targeting these DDR pathways to improve therapeutic outcomes. More recently, we have adopted dTAG technology for the investigation of many essential DDR and replication proteins, which provide mechanistic insights into the roles of these essential proteins in genome maintenance. Additionally, we have expanded FACS-based screens for the studies of other cancer-associated pathways and explored key biological processes critical for cancer progression in vivo. These studies will facilitate the development of better therapies for cancer patients. Citation Format: Junjie Chen. Targeting DNA damage responsive pathways in cancer therapy. [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Translating Targeted Therapies in Combination with Radiotherapy; 2025 Jan 26-29; San Diego, CA. Philadelphia (PA): AACR; Clin Cancer Res 2025;31(2_Suppl):Abstract nr A009.

The fidelity of the genome is under constant threat by exogenous and endogenous reactive species, including toxic chemicals, ionizing radiation and byproducts of normal cellular metabolism. These species cause damage to DNA by modifying DNA bases, breaking DNA strands and/or altering DNA structures. When this happens, the consequences to the cell can be disastrous, ranging from single gene mutations to massive chromosomal breakdown and rearrangements. These instabilities lead to severe human diseases including cancer. Fortunately, humans and other eukaryotes have evolved multiple mechanisms to ensure genome stability. These mechanisms can be classified into two major systems, one that deals with replication fidelity and the other that takes care of various DNA lesions. The replication fidelity maintenance system involves DNA polymerases and the DNA mismatch repair pathway. First, the 3'-5' proofreading nuclease activity of replicative DNA polymerases is responsible for removing the majority of mis-incorporated nucleotides during DNA replication [1]. However, there are always cases where certain mis-incorporations escape from the proofreading activity. In the latter situation, the mismatch repair machinery [2] is recruited to the site to correct the mismatch. If a DNA template contains a bulky DNA adduct that blocks the polymerization activity of replicative polymerases, a specialized DNA polymerase, also called translesion synthesis polymerase, is employed to bypass the bulky DNA lesion, followed by a switch back to the replicative polymerase to continue normal replication [3]. The DNA damage processing system can be divided into two steps: DNA damage response and DNA repair. Extensive studies in the past 20 years have elucidated the mechanisms of several major DNA repair pathways that remove almost all kinds of DNA lesions. These pathways include base excision repair [4], nucleotide excision repair [5,6], mismatch repair [2] and double strand break repair [7,8]. DNA damage response is a complex signal transduction process that has the ability to sense DNA damage and transduce the information to the cell to direct cellular responses to the damage. An arsenal of protein activities has been identified, which function to be damage sensors, mediators, transducers and effectors during DNA damage response [9]. Unlike the DNA repair pathways, however, the mechanism of DNA damage response is much less known. In this issue, Cell & Bioscience presents a series of reviews attempting to define the most challenging questions in DNA damage response and repair, and to provide an overview of the latest breakthroughs and developments in the field. The article by Nan Wu and Hongtao Yu [10] explores the mechanism by which the structural maintenance of chromosomes (Smc) proteins and non-Smc proteins, which are required for chromatid cohesion, chromosomal segregation and condensation, regulate DNA damage response and repair. A second article by Bin Wang [11] describes how BRCA1, an important tumor suppressor and a critical DNA damage response mediator, interacts with its partners to regulate activities of multiple repair and checkpoint pathways for genome maintenance. Maintaining the stability of repetitive DNA sequences, particularly the (CAG)n and (CTG)n trinucleotide repeats, whose expansions cause numerous human disorders, is extremely challenging, as is the understanding of the mechanisms that regulate the stability/instability of the repetitive sequences. In their article, Guoqi Liu and Michael Leffak [12] debate how collaborative efforts by the replication machinery and multiple DNA repair pathways stabilize (CAG)n and (CTG)n trinucleotide repeats. It is our sincere hope that this special issue brings our readers enlightenment and offers sufficient introductory information to help them appreciate new breakthroughs in the field.

Maintenance of genomic integrity is one of the fundamental biological properties shared by all living organisms. To counterbalance deleterious and potentially mutagenic effects of omnipresent DNA damaging assaults, organisms have developed a network of genome surveillance and maintenance pathways known as the DNA damage response. In eukaryotes, the orchestration of cell-cycle checkpoints, DNA damage repair, and apoptosis in response to DNA damage relies on posttranslational modifications of key regulatory proteins. Although the role of phosphorylation in these pathways is relatively well established, the significance of ubiquitylation has only recently emerged. In this review, we survey current research on the ubiquitin-proteasome system, focusing on the DNA damage response in the G2 phase of the cell cycle and two prominent classes of ubiquitin ligases, the SCF- and APC/C complexes. These ubiquitin ligases are reviewed with regard to their function in activating, maintaining, and terminating the checkpoint and in light of increasing evidence that suggests a dynamic balance of substrate ubiquitylation and deubiquitylation. We further discuss the impact of defective G2 checkpoint signaling on genomic stability and cancer risk, highlighting strategies for targeted antitumor drug discovery.

Genome maintenance is an enabling characteristic that allows neoplastic cells to tolerate the inherent stresses of tumorigenesis and evade therapy-induced genotoxicity. Neoplastic cells also deploy many mis-expressed germ cell proteins termed Cancer Testes Antigens (CTAs) to promote genome maintenance and survival. Here, we present the first comprehensive characterization of the DNA Damage Response (DDR) and CTA transcriptional landscapes of endometrial cancer in relation to conventional histological and molecular subtypes. We show endometrial serous carcinoma (ESC), an aggressive endometrial cancer subtype, is defined by gene expression signatures comprising members of the Replication Fork Protection Complex (RFPC) and Fanconi Anemia (FA) pathway and CTAs with mitotic functions. DDR and CTA-based profiling also defines a subset of highly aggressive endometrioid endometrial carcinomas (EEC) with poor clinical outcomes that share similar profiles to ESC yet have distinct characteristics based on conventional histological and genomic features. Using an unbiased CRISPR-based genetic screen and a candidate gene approach, we confirm that DDR and CTA genes that constitute the ESC and related EEC gene signatures are required for proliferation and therapy-resistance of cultured endometrial cancer cells. Our study validates the use of DDR and CTA-based tumor classifiers and reveals new vulnerabilities of aggressive endometrial cancer where none currently exist.

Microtubules are major components of the cytoskeleton that play important roles in cellular processes such as intracellular transport and cell division. In recent years, it has become evident that microtubule networks play a role in genome maintenance during interphase. In this review, we highlight recent advances in understanding the role of microtubule dynamics in DNA damage response and repair. We first describe how DNA damage checkpoints regulate microtubule organization and stability. We then highlight how microtubule networks are involved in the nuclear remodeling following DNA damage, which leads to changes in chromosome organization. Lastly, we discuss how microtubule dynamics participate in the mobility of damaged DNA and promote consequent DNA repair. Together, the literature indicates the importance of microtubule dynamics in genome organization and stability during interphase.

Single-stranded DNA-binding protein in Bacteria and replication protein A (RPA) in Eukarya play crucial roles in DNA replication, repair, and recombination processes. We identified an RPA complex from the hyperthermophilic archaeon, Pyrococcus furiosus. Unlike the single-peptide RPAs from the methanogenic archaea, Methanococcus jannaschii and Methanothermobacter thermoautotrophicus, P. furiosus RPA (PfuRPA) exists as a stable hetero-oligomeric complex consisting of three subunits, RPA41, RPA14, and RPA32. The amino acid sequence of RPA41 has some similarity to those of the eukaryotic RPA70 subunit and the M. jannaschii RPA. On the other hand, RPA14 and RPA32 do not share homology with any known open reading frames from Bacteria and Eukarya. However, six of eight archaea, whose total genome sequences have been published, have the open reading frame homologous to RPA32. The PfuRPA complex, but not each subunit alone, specifically bound to a single-stranded DNA and clearly enhanced the efficiency of an in vitro strand-exchange reaction by the P. furiosus RadA protein. Moreover, immunoprecipitation analyses showed that PfuRPA interacts with the recombination proteins, RadA and Hjc, as well as replication proteins, DNA polymerases, primase, proliferating cell nuclear antigen, and replication factor C in P. furiosus cells. These results indicate that PfuRPA plays important roles in the homologous DNA recombination in P. furiosus.

Studies on cancer-prone and rare human genetic disorders often lead to significant advances in our understanding of the complex network of genome stability and DNA repair pathways that have evolved in the human genome to prevent the harmful effects of exposure to DNA damaging agents. One such disorder is Fanconi Anemia, an autosomal recessive disease characterized by an increased spontaneous and DNA cross-linkers induced chromosome instability, progressive pancytopenia and cancer susceptibility. At least eleven genes are involved in Fanconi anemia, including the breast cancer susceptibility gene BRCA2. Six of the Fanconi anemia proteins (FANCA, C, E, F, G and L) assemble in a complex that is required for FANCD2 activation by monoubiquitination in response to DNA damage or during S-phase progression. Active FANCD2 then colocalizes with the product of the breast cancer susceptibility gene BRCA1 in discrete nuclear foci. FANCD2 is also independently phosphorylated by ATM in response to ionising radiation and interacts with the MRE1 l/Rad50/NBSl complex, which is directly involved in homologous recombination DNA repair pathway and in cell cycle checkpoint response to DNA damage. Available data indicate that FANCD2 is involved in cell cycle regulation and DNA repair. Our current knowledge on the functional significance of FA pathway and more specifically FANCD2 and its interacting proteins in pathways of genomic surveillance and maintenance will be discussed in this chapter.

Function and molecular mechanisms of APE2 in genome and epigenome integrity

In addition to its role in chromosome maintenance, the six-membered Smc5/6 complex functions as a restriction factor that binds to and transcriptionally silences viral and other episomal DNA. However, the underlying mechanism is unknown. Here, we show that transcriptional silencing by the human Smc5/6 complex is a three-step process. The first step is entrapment of the episomal DNA by a mechanism dependent on Smc5/6 ATPase activity and a function of its Nse4a subunit for which the Nse4b paralog cannot substitute. The second step results in Smc5/6 recruitment to promyelocytic leukemia nuclear bodies by SLF2 (the human ortholog of Nse6). The third step promotes silencing through a mechanism requiring Nse2 but not its SUMO ligase activity. By contrast, the related cohesin and condensin complexes fail to bind to or silence episomal DNA, indicating a property unique to Smc5/6.

Besides being a mediator of initiation of DNA replication, DNA polymerase α plays a key role in chromosome maintenance. Swi7H4, a novel temperature sensitive mutant of DNA polymerase α was shown to be defective in transcriptional silencing at the mating type centromere and telomere loci. It is also required for the establishment of chromatin state that can recruit the components of the heterochromatin machinery at these regions. Recently the role of DNA polymerase α in the S-phase alkylation damage response in S. pombe has also been studied. Here we investigate whether defects generated by swi7H4, a mutant allele of DNA polymerase α can activate a checkpoint response. We show that swi7H4 exhibit conditional synthetic lethality with chk1 null mutant and the double mutant of swi7H4 with chk1 deletion aggravate the chromosome segregation defects. More importantly swi7H4 mutant cells delay the mitotic progression at non permissive temperature that is mediated by checkpoint protein kinase Chk1. In addition we show that, in the swi7H4 mutant background, cells accumulate DNA damage at non permissive temperature activating the checkpoint kinase protein Chk1. Further, we observed synthetic lethality between swi7H4 and a number of genes involved in DNA repair pathway at semi permissive temperature. We summarize that defects in swi7H4 mutant results in DNA damage that delay mitosis in a Chk1 dependent manner that also require the damage repair pathway for proper recovery.

The functional roles of PML nuclear bodies in genome maintenance

Single Stranded DNA Translocases can Push Single Stranded DNA Binding Proteins along Single Stranded DNA