title: Delineating the overlapping roles of the single-stranded DNA binding proteins Ssb1 and Ssb2 in the maintenance of genomic stability and intestinal homeostasis
Creators: name: Nag, Purba
Publisher: name: Griffith University
Year: 2019
Subjects: subject: Single-stranded DNA
subject: Binding proteins (SSBPs)
subject: DNA damage response
subject: Genomic stability
subject: Molecular mechanisms

description: 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.