1. 160805
WOORI BAE

2. R-loops and genome integrity
Model for processing of R-loops into double-strand breaks (DSBs)
Physiological roles of R-loops in cells.
(B) The endonucleases XPF and XPG can recognize both strands of an R-loop and generate a DSB. (C) The TC-NER machinery may be recruited to R-loops during replication, and the concerted action of the endonucleases XPF and XPG with other structure-specific endonucleases may generate a DSB.
(A) The endonucleases XPF and XPG can excise the RNA–DNA hybrid of an R-loop. The single-stranded gap may be converted into a DSB either by the action of structure-specific endonucleases, or by encounter with the replication fork, which can also involve structure-specific endonucleases.
Trends in Cell Biology September 2015

3. Persistent R-loop formation and consequential collision with DNA and RNA synthetic processes result in complex DSBs
Specific DNA lesions and/or loss of specific RNA processing factors, such as RNA transcription termination factors, can result in the formation of persistent R-loops. RNA polymerase II (RNA Pol II or RNAPII, text) proceeds along its DNA coding template, releasing the mRNA for poly(A) processing. At the RNA transcriptional termination sequence, RNAPII stalls awaiting a specific sequence of RNA transcription termination factors. K-H and p15RS are essential. If K-H, p15RS or a host of other factors are not present, RNA Pol II (RNAPII) fails to stall and a persistent DNA:RNA:DNA structure (an R-loop) is formed. This structure is hypersensitive to secondary damage or mis-repair from nucleotide excision repair (NER) or base excision repair (BER).
Persistent R-loops represent a barrier to RNA or DNA synthetic forks, creating collisions that create even more complex DSBs, which can be repaired by Artemis (Morales et al., 2014).
Julio C. Morales et al., 2015

4. R-loops are associated with chromatin marks.
R-loops influence both transcriptional initiation and termination. R-loops located at promoters prevent DNA methylation, which leads to the establishment of active chromatin marks and transcriptional initiation.
By contrast, R-loops located at termination regions induce the formation of heterochromatin promoting transcriptional termination.
In addition, the RNA/DNA helicase SETX resolves R-loops at termination regions to promote transcriptional termination.

5. Kub5-Hera (K-H) is an essential scaffolding protein required for RNA transcription termination, RNA transcription and DSB repair complex formation
(A) K-H is an essential protein for the functional assembly of RNA termination factors to dislodge RNA Pol II (RNAPII) and mediate XRN2 degradation of the RNA component of an R-loop, thereby resolving persistent R-loops (Adapted from Richard, Genes and Dev., 2009). (B) K-H forms a complex with specific non-homologous end joining recombination factors and is required for stabilization of Artemis to mediate repair of complex DSBs (Morales, NAR, 2014)
Julio C. Morales et al., 2015

6. The torpedo model for transcription termination by RNA polymerase II_XRN2
pre-mRNA is subjected to a series of covalent modifications during its synthesis: capping, splicing, cleavage at the poly(A) site, polyadenylation, cleavage at the CoTC site, 5 ′→ 3 ′ degradation of downstream RNA and degradation of the RNA by-product between the poly(A) and CoTC sites.
CoTC is a ribozyme, 5 ′→ 3 ′ degradation of downstream RNA is carried out by Xrn2 exonuclease and both processes are required for termination. 
Nature Structural & Molecular Biology, 2005

7. Figure 1. XRN2 undergoes DNA damage-inducible nuclear re-localization: R-loop formation and active transcription are both required for XRN2 foci formation after genomic insult
whether XRN2 associated with known DNA repair factors
gel filtration chromatography
- XRN2 and several DDR factors interact with SETX
- BRCA1 mediates SETX recruitment
- Ku80 has also been found to interact with SETX in
an affinity purification of FLAG-tagged SETX
NHEJ pathway
XRN2 foci also co-localized with R-loop foci
whether XRN2 formed DNA damage-induced foci
S9.6 antibody: recognizes RNA:DNA hybrids
shRNA control (shScr) or a Kub5-Hera shRNA (shk-h) in mock (unt)
RNAPII inhibitor α-amanitin

8. Figure 2. Loss of XRN2 results in increased DSBs and genomic instability
Nucleic Acids Research. 2014: loss of K-H and p54(nrb), two factors implicated in mediating XRN2 genomic distribution, led to increased DSB formation
shXRN2: increased level of basal DNA damage
similar levels of chromosome-type damage, but loss of XRN2 led to significantly more chromatid-type damage, which was not seen in K-H deficient cells- important difference between the two transcription termination factors

9. Figure 3. Loss of XRN2 sensitizes cells to a wide variety of genomic insult
hypersensitive to various genomic insults
decreased colony forming ability after exposure to IR, aphidicolin (APH) or hydrogen peroxide (H2O2), UV radiation
difference between XRN2- and SETX-deficient cells, which show sensitivity to oxidative DNA damage but not IR
difference between XRN2 and K-H deficient cells, which are sensitive to IR, but not UV

10. Figure 4. Cells lacking XRN2 undergo increased replication stress
PCNA, a marker of cells undergoing DNA synthesis
increased phosphorylation of RPA32, activated ATR, and the checkpoint kinase CHK1
increased amounts of chromatid damage
accumulation of several factors required for homologous recombination
loss of XRN2 can cause replicative stress
replication fork impairment: XRN2-deficient cells undergo significantly increased replication stress

11. Figure 5. Loss of XRN2 results in increased R-loop formation
Excess R loop formation
R loops that form in the nucleus tend to be sensitive to RNaseH, while R loops within the nucleolus tend to be RNaseH resistant
XRN2 in R-loop removal and protection from DSB accumulation
the S9.6 signal was strongly diminished after RNaseH

12. Figure 6. R loop formation inhibits DNA repair after genomic insult
the effects of IR on R-loop formation in shXRN2 cells
R loops can directly lead to DSBs
whether active transcription or R-loop formation directly affected DNA repair (regression of 53BP1 foci) after IR treatment
inhibition of transcription: α-amanitin
removal of R loops: RNaseH
completely restored DNA repair kinetics after IR exposure
XRN2-deficient cells, along with an inability to properly perform DSB repair through the NHEJ pathway, are defective in R-loop resolution after IR

13. Figure 7. Loss of XRN2 leads to accumulation of DDR proteins at 3’ transcriptional pause sites of genes
Whether DDR factors accumulate at transcriptional pause sites on genes that undergo R-loop-dependent termination in XRN2-depleted cells
ENSA: highly conserved cAMP-regulated phosphoprotein (ARPP) family
Gemin: survival of motor neuron protein
Akirin: embryonic development and for normal innate immune response
Int: Intronic region of Gemin7
no change
XRN2 plays an important role in maintaining genomic integrity at the 3’ pause sites of genes
no enrichment

14. SUMMARY_Model for XRN2 functions in DNA repair pathway choice
In normal conditions,
XRN2 binds to the NHEJ factor 53BP1 promoting DSB repair via the NHEJ pathway.
In the absence of XRN2,
NHEJ is inhibited downstream of 53BP1, allowing DSB repair via
the HR pathway.