Vanderbilt Institute of Chemical Biology



Discovery at the VICB







Guarding the Genome with SMARCAL1


By: Carol A. Rouzer, VICB Communications
Published:  November 20, 2015



The replication stress response protein SMARCAL1 plays an important role in the normal, but challenging, replication of telomeric DNA.


Cells face enormous challenges during the process of DNA replication. Not only must they faithfully replicate the billions of base pairs in the genome, they must do so in the context of multiple stressors, including damaged bases, sequences that form odd structures, and interference caused by transcription machinery. To cope with these impediments, cells have evolved numerous mechanisms to repair damage and to resolve and restart stalled replication forks. Among the stress response proteins is a group of DNA translocases that perform branch migration reactions using the energy of ATP hydrolysis. This group includes SMARCAL1 (SWI/SNF-related, matrix-associated, actin-dependent, regulator of chromatin, subfamily A-like 1), ZRANB3 (zinc finger, RNA-binding domain-containing 3), and HLTF (helicase-like transcription factor). All of these enzymes catalyze branch migration in vitro, and both SMARCAL1 and ZRANB3 also catalyze DNA strand annealing, disrupt displacement loop structures, and restore replication forks. Gene knockdown studies in cells have revealed subtle functional differences between the translocases, particularly with regard to their roles in protecting cells against exogenously added genotoxic stressors (e.g., UV light, methylating agents, cross-linking agents). Yet, despite extensive study, the exact role for each of these proteins in the response to DNA replication stress remains unclear. This is especially true in the case of stress that occurs during normal replication, in the absence of exogenous toxicants. Now, Vanderbilt Institute of Chemical Biology member David Cortez and his laboratory show that SMARCAL1 plays a unique role in the replication of telomeric DNA [L. A. Poole, et al., (2015) Proc. Natl. Acad. Sci. U.S.A., published online November 17, DOI: 10.1073/pnas.1510750112].


SMARCAL1 is a multi-domain protein comprising an ATPase catalytic domain, a HARP1 domain that plays a role in DNA remodeling, and an N-terminal replication protein A- (RPA)-binding domain (Figure 1). Its ability to bind to RPA facilitates localization of SMARCAL1 to replication forks, and the RPA interaction also modulates SMARCAL1 activity. Inherited mutations in the gene for SMARCAL1 lead to the recessive genetic disorder Schimke immunoosseous dysplasia (SIOD), a disease associated with renal failure, growth defects, immune deficiencies, and a predisposition to cancer. Mutations associated with SIOD have been identified across the entire gene for SMARCAL1 (Figure 1). All of these features distinguish SMARCAL1 from ZRANB3 and HLTF, but an even more important distinction for the Cortez lab investigators was the prior finding that knockdown of SMARCAL1 leads to increased DNA damage in cells not exposed to any external stressors. This observation suggested the hypothesis that SMARCAL1 likely plays a role in the response to endogenous replication stress.


Figure 1. Domain structure of SMARCAL1. The ATPase catalytic domain is designated by the gold bar. It includes two RecA ATPase domains (dark blue and red), two SNF2-specific helical domains (light blue and pink), and a HARP2 DNA binding domain (green). Other domains include the replication protein A-binding domain (RBD), located at the N-terminus, and the HARP1 domain, which plays a role in DNA remodeling. The x and o markings indicate the sites of mutations associated with SIOD. Figure reproduced by permission from A. C. Mason, et. al., (2001) Proc. Natl. Acad. Sci. U.S.A., 111, 7618. Copyright 2014 A. C. Mason, et. al.


To test this hypothesis, the investigators focused on replication of the telomeric DNA found at the terminus of all chromosomes. In vertebrates, telomeres comprise long (up to 15 kb) stretches of repeated 5′-TTAGGG-3′/3′-AATCCC-5′ sequences (Figure 2). The repetitive sequence allows the DNA to form physiological t-loop structures, but it also enables the formation of aberrant structures that can lead to replication stress. The potential damage this can cause is exacerbated by the fact that telomeric DNA contains few origins of replication, so if a replication fork becomes stalled, there are limited opportunities to restart the process.



Figure 2. Structure of the vertebrate telomere. (a) The telomere comprises 5´TTAGGG-3´/3´-AATCCC-5´ repeats of 4 to 14 kb in length (in normal human cells). The end of the 5´-TTAGGG-3´ strand is extended for 130-230 nucleotides as a single-stranded tail. (b) The telomere may form a t-loop structure with the single-stranded tail displacing a region of the duplex to pair with its complementary 3´-AATCCC-5´ strand. (c & d) Both the linear and looped structures are stabilized and protected by a coating of shelterin complex proteins. The shelterin complex includes TRF1 and TRF2, which bind directly to the DNA duplex, POT1, which binds to the single-stranded tail, and TPP1, TIN2, and RAP1, which interact with the DNA indirectly by binding to the other proteins. Figure reproduced by permission from Macmillan Publishers, Ltd. from A. J. Cesare & R. R. Reddel, (2001) Nat. Rev. Gen., 11, 319. Copyright 2010.



To determine if SMARCAL1, plays a role in ensuring efficient replication of telomeric DNA, the Cortez lab investigators used siRNA to knockdown expression of the protein in HeLa1.3 cells, followed by fluorescence microscopy to search for evidence of DNA damage at telomeres during S phase of the cell cycle. Their results (Figure 3) showed that, indeed, SMARCAL1 knockdown led to an accumulation of the DNA damage response proteins 53BP1 and RPA at sites enriched with the 5′-TTAGGG-3′ sequence. They designated these sites telomere dysfunction-induced foci (TIFs). The increase in TIFs was abrogated when the researchers transfected SMARCAL1 siRNA-treated cells with an siRNA-resistant gene encoding wild-type SMARCAL1. Rather unexpectedly, transfection with a gene encoding an N-terminal deletion mutant of SMARCAL also reduced the TIF increase observed in SMARCAL1 siRNA-treated cells. These findings suggested that SMARCAL1 is required for normal replication of telomeric DNA. However, they also indicated that SMARCAL1 does not need to interact with RPA to execute this function, as the N-terminal deletion mutant lacked the RPA-binding domain.




Figure 3. SMARCAL1 is required for successful telomere replication. HeLa1.3 cells were transfected with siRNA directed against SMARCAL1 (siSM1-1) or with nontargeting siRNA (siNT). The cells were then stained with DAPI to label the nuclei, a fluorescent antibody against 53BP1 (a DNA damage response protein), and a fluorescent telomeric DNA probe (TTAGGG). Fluorescence microscopy revealed that knockdown of SMARCAL1 led to an increase in 53BP1-containing foci (green), indicating an increase in sites of DNA damage. A significant number of these colocalized with telomeric DNA sequences (red), as indicated by the merged images (arrows). Reproduced with permission from L. A. Poole, et al., (2015) Proc. Natl. Acad. Sci. U.S.A., published online November 17, DOI: 10.1073/pnas.1510750112. Copyright 2015 L. A. Poole, et al.



Abnormal replication of telomeres can lead to the formation of C-circles, extrachromosomal, partially duplexed circular DNA fragments. The Cortez lab demonstrated that siRNA-mediated knockdown of SMARCAL1 resulted in an increase in C-circle formation in HeLa1.3 cells. They also found elevated levels of C-circles in mouse embryo fibroblasts (MEFs) derived from Smarcal1Δ/Δ knockout mice as compared to those of MEFs from wild-type mice. Expression of wild-type human SMARCAL1 in the knockout MEFs restored the level of C-circle formation to that of wild-type cells, as did expression of the N-terminal deletion mutant of SMARCAL1. However, expression of a mutant SMARCAL1 obtained from an SIOD patient had no effect on C-circle formation in Smarcal1Δ/Δ MEFs. The SIOD mutant protein lacked ATPase activity, suggesting that this catalytic function is required for SMARCAL1’s role in promoting telomere replication.


C-circle formation is associated with alternative lengthening of telomeres (ALT), an abnormal process frequently found in cancer cells. This led the Cortez lab to hypothesize that a deficiency of SMARCAL1 might lead to an increase in ALT-dependent processing of telomeres. However, they found no evidence of other alterations of telomere structure, such as excessive length, variability in length, or increase in the presence of promyelocytic leukemia protein, that usually accompany ALT. Thus, they concluded that the effect of SMARCAL1 deficiency on C-circle formation happens independently of ALT. However, they also discovered that formation of C-circles in SMARCAL1 deficient cells requires the SLX4 nuclease scaffold protein, a characteristic shared with C-circle formation in ALT.


The apparently important role of SMARCAL1 in telomere replication led the investigators to hypothesize that SMARCAL1 should be detectable in telomeres during S phase. However, when they tested this hypothesis, they discovered that they could only detect telomere-associated SMARCAL1 when they overexpressed the protein at levels high enough to induce DNA damage. This finding did not support a role for SMARCAL1 in telomere replication; however, it is possible that the association of SMARCAL1 with telomeric DNA is transient, happening too quickly to be detectable by the methods employed.


Together, the results suggest that SMARCAL1 modulates telomere replication under normal physiological conditions. This role requires SMARCAL1’s ATPase catalytic activity, but not its ability to bind to RPA. The latter finding is, perhaps, not a surprise, as RPA is generally not abundant in telomeric DNA. Likely, SMARCAL1 interacts with telomeric DNA directly through its high affinity DNA binding sites that can function independently of RPA. This is the first reported role for SMARCAL1 in the resolution of an endogenous replication stress. Furthermore, as knockdown of HLTF or ZRANB3 had no effect on telomere replication, this appears to be a unique function of SMARCAL1. This discovery provides important new information on how the cell maintains genomic integrity in the face of what would otherwise be insurmountable chemical and physical challenges.



View PNAS article: SMARCAL1 maintains telomere integrity during DNA replication








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