We identified SMARCAL1 in two functional genetic screens designed to identify novel genome maintenance activities in human cells.14
The rationale was as follows: deregulation of a genome maintenance activity by either RNAi depletion or cDNA overexpression will result in an increase in spontaneous DNA damage without the addition of genotoxic agents; the increase in DNA damage burden can be monitored by detecting the phosphorylation status of DNA damage response substrates. Both SMARCAL1 silencing and overexpression activate the DNA damage response specifically in replicating cells.5
In addition, silencing SMARCAL1 renders cells hypersensitive to replication stress agents like hydroxyurea (HU), camptothecin and aphidicolin.5,6
These data suggest that SMARCAL1 activity is critical for genome maintenance during DNA replication.
When DNA lesions persist or occur during S phase, they can cause uncoupling of helicase and polymerase activities due to polymerase stalling.15
This functional uncoupling generates significant amounts of single-stranded DNA (ssDNA) as the helicase continues to unwind double-stranded DNA. The ssDNA binding protein, replication protein A (RPA), coats the DNA and recruits multiple replication stress response proteins to the stalled replication fork.16,17
Following replication stress, SMARCAL1 localizes to sites of stalled replication, co-localizing with RPA and other markers of stalled forks.5–7,9
The N-terminus of SMARCAL1 has sequence similarity to an RPA-binding region in the replication stress response protein TIPIN (Timeless interacting protein).18
Similar to TIPIN, this region of SMARCAL1 interacts with the 32 kDa subunit of RPA (RPA2).5,6
The RPA-interaction domain of SMARCAL1 is both necessary and sufficient to localize SMARCAL1 to stalled replication forks.5
Importantly, a SMARCAL1 mutant (ΔN) that does not bind RPA cannot rescue the replication-associated DNA damage observed in SMARCAL1-deficient cells.5,9
The ΔN-SMARCAL1 mutant still binds forked DNA in vitro and retains DNA stimulated ATPase activity and annealing helicase activity.5,13
Although the RPA interaction is not essential for SMARCAL1 enzymatic activities in vitro, the genome maintenance activities of SMARCAL1 in cells depend upon its interaction with RPA. It is likely that RPA functions to localize SMARCAL1 to its substrate at the replication fork.
If SMARCAL1 functions as an annealing helicase at stalled forks then we would expect to observe more ssDNA in SMARCAL1-silenced cells. Indeed, silencing SMARCAL1 leads to an increase in ssDNA especially in the presence of replication stress.5,9
Furthermore, SMARCAL1-silenced cells recover more slowly after resolution of the stalled fork since damage-dependent RPA phosphorylation persists in SMARCAL1-silenced cells after removing replication stress-inducing agents from the growth media,5
and fiber labeling indicates replication fork restart is slower.6
These results are consistent with a model in which SMARCAL1 anneals ssDNA at stalled forks, reduces the amount of ssDNA available for RPA binding, and promotes fork stability.
Why would cells require ssDNA annealing at stalled forks? The DNA at a replication fork is nucleosome-free and may have an increased propensity to form bubbles. Furthermore the 3′ and 5′ ends of nascent DNA strands could unwind from the template and form flaps especially during the process of polymerase switching at a DNA lesion. SMARCAL1 could function to reduce bubble and flap formation, an activity that would prevent inappropriate processing of the DNA. SMARCAL1 could also limit uncoupling of polymerase and the MCM helicase functioning as an anti-helicase directly at the fork to reduce ssDNA. Certainly other models are possible19
so further analysis of SMARCAL1 biochemical activities including the identification of its substrates will be essential to fully understand its genome maintenance activity.