Co-transcriptional R-loops are the main cause of estrogen-induced DNA damage.

The hormone estrogen (E2) binds the estrogen receptor to promote transcription of E2-responsive genes in the breast and other tissues. E2 also has links to genomic instability, and elevated E2 levels are tied to breast cancer. Here, we show that E2 stimulation causes a rapid, global increase in the formation of R-loops, co-transcriptional RNA-DNA products, which in some instances have been linked to DNA damage. We show that E2-dependent R-loop formation and breast cancer rearrangements are highly enriched at E2-responsive genomic loci and that E2 induces DNA replication-dependent double-strand breaks (DSBs). Strikingly, many DSBs that accumulate in response to E2 are R-loop dependent. Thus, R-loops resulting from the E2 transcriptional response are a significant source of DNA damage. This work reveals a novel mechanism by which E2 stimulation leads to genomic instability and highlights how transcriptional programs play an important role in shaping the genomic landscape of DNA damage susceptibility

Continued primer synthesis at stalled replication forks contributes to checkpoint activation

An increased number of primer–template junctions generated by PCNA, Pol-δ, and Pol-ε at stalled replication forks activates Chk1

HARPing on about the DNA damage response during replication

In this issue of Genes & Development, four papers report that the annealing helicase HepA-related protein (HARP, also known as SMARCAL1 [SWI/SNF-related, matrix-associated, actin-dependent regulator of chromatin, subfamily a-like 1]) binds directly to the ssDNA-binding protein Replication protein A (RPA) and is recruited to sites of replicative stress. Knockdown of HARP results in hypersensitivity to multiple DNA-damaging agents and defects in fork stability or restart. These exciting insights reveal a key new player in the S-phase DNA damage response

Checkpoint recovery after DNA damage: a rolling stop for CDKs

The cell cycle machinery has to be arrested for efficient repair of DNA damage. A paper in this issue of EMBO reports demonstrates that residual CDK activity is nevertheless required during arrest for activation of FoxM1, which is essential for cell-cycle progression after the damage has been repaired

The structural determinants of checkpoint activation

Here, we demonstrate that primed, single-stranded DNA (ssDNA) is sufficient for activation of the ATR-dependent checkpoint pathway in Xenopus egg extracts. Using this structure, we define the contribution of the 5′- and 3′-primer ends to Chk1 activation when replication is blocked and ongoing. In addition, we show that although ssDNA is not sufficient for checkpoint activation, the amount of ssDNA adjacent to the primer influences the level of Chk1 phosphorylation. These observations define the minimal DNA requirements for checkpoint activation and suggest that primed ssDNA represents a common checkpoint activating-structure formed following many types of damage

A requirement for replication in activation of the ATR-dependent DNA damage checkpoint

Using the Xenopus egg extract system, we investigated the involvement of DNA replication in activation of the DNA damage checkpoint. We show here that DNA damage slows replication in a checkpoint-independent manner and is accompanied by replication-dependent recruitment of ATR and Rad1 to chromatin. We also find that the replication proteins RPA and Polα accumulate on chromatin following DNA damage. Finally, damage-induced Chk1 phosphorylation and checkpoint arrest are abrogated when replication is inhibited. These data indicate that replication is required for activation of the DNA damage checkpoint and suggest a unifying model for ATR activation by diverse lesions during S phase

Functional uncoupling of MCM helicase and DNA polymerase activities activates the ATR-dependent checkpoint

The ATR-dependent DNA damage response pathway can respond to a diverse group of lesions as well as inhibitors of DNA replication. Using the Xenopus egg extract system, we show that lesions induced by UV irradiation and cis-platinum cause the functional uncoupling of MCM helicase and DNA polymerase activities, an event previously shown for aphidicolin. Inhibition of uncoupling during elongation with inhibitors of MCM7 or Cdc45, a putative helicase cofactor, results in abrogation of Chk1 phosphorylation, indicating that uncoupling is necessary for activation of the checkpoint. However, uncoupling is not sufficient for checkpoint activation, and DNA synthesis by Polα is also required. Finally, using plasmids of varying size, we demonstrate that all of the unwound DNA generated at a stalled replication fork can contribute to the level of Chk1 phosphorylation, suggesting that uncoupling amplifies checkpoint signaling at each individual replication fork. Taken together, these observations indicate that functional uncoupling of MCM helicase and DNA polymerase activities occurs in response to multiple forms of DNA damage and that there is a general mechanism for generation of the checkpoint-activating signal following DNA damage

Proliferating Cell Nuclear Antigen Uses Two Distinct Modes to Move along DNA

Proliferating cell nuclear antigen (PCNA) plays an important role in eukaryotic genomic maintenance by topologically binding DNA and recruiting replication and repair proteins. The ring-shaped protein forms a closed circle around double-stranded DNA and is able to move along the DNA in a random walk. The molecular nature of this diffusion process is poorly understood. We use single-molecule imaging to visualize the movement of individual, fluorescently labeled PCNA molecules along stretched DNA. Measurements of diffusional properties as a function of viscosity and protein size suggest that PCNA moves along DNA using two different sliding modes. Most of the time, the clamp moves while rotationally tracking the helical pitch of the DNA duplex. In a less frequently used second mode of diffusion, the movement of the protein is uncoupled from the helical pitch, and the clamp diffuses at much higher rates