Poly(ADP-ribose) binds to the splicing factor ASF/SF2 and regulates its phosphorylation by DNA topoisomerase I
Authors: Felix R Althaus
Maria Malanga
Agnieszka Girstun
Krzysztof Staron
Alicja Czubaty
Journal: Journal of Biological Chemistry (2008)
Discipline: BIOLOGY & BIOCHEMISTRY
Keywords: DNA topoisomerase I; transcription; DNA supercoiling; kinase; SR-protein family
Affiliations: Department of Structural and Functional Biology, University Federico II, Via Cinthia, Monte S Angelo, Naples, Italy. 

Abstract:  Human DNA topoisomerase I plays a dual role in transcription, by controlling DNA supercoiling and by acting as a specific kinase for the SR-protein family of splicing factors. The two activities are mutually exclusive, but the identity of the molecular switch is unknown. Here we identify poly(ADP-ribose) as a physiological regulator of the two topoisomerase I functions. We found that, in the presence of both DNA and the alternative splicing factor/splicing factor 2 (ASF/SF2, a prototypical SR-protein), poly(ADP-ribose) affected topoisomerase I substrate selection and gradually shifted enzyme activity from protein phosphorylation to DNA cleavage. A likely mechanistic explanation was offered by the discovery that poly(ADP-ribose) forms a high affinity complex with ASF/SF2 thereby leaving topoisomerase I available for directing its action onto DNA. We identified two functionally important domains, RRM1 and RS, as specific poly(ADP-ribose) binding targets. Two independent lines of evidence emphasize the potential biological relevance of our findings: (i) in HeLa nuclear extracts, ASF/SF2, but not histone, phosphorylation was inhibited by poly(ADP-ribose); (ii) an in silico study based on gene expression profiling data revealed an increased incidence of alternative splicing within a subset of inflammatory response genes that are dysregulated in cells lacking a functional poly(ADP-ribose) polymerase-1. We propose that poly(ADP-ribose) targeting of topoisomerase I and ASF/SF2 functions may participate in the regulation of gene expression. 

Retracted: No

PubPeer comments:
 Figure 5B appears to have a band that has been reused, after adjusting brightness contrast and adjusting dimensions. Could the authors please provide the original uncropped scans to dismiss any concerns? *** ![file] , 
 Thank you very much for deeply analyzing our control blots. The two bands given for p38 and GAPDH are definitely different and are generated from two different blots probed with anti-p38 and anti-GAPDH respectively. Given above are the two blots. Blot 1 is for p38. Blot 2 is for GAPDH. Since both the bands come on similar size and both the gels were loaded with the same amount of cell lysates, hence the similarities. However, p38 gel is not straight. In the process of making the final images with same size and width, the blots were stretched a little bit, which makes two blots alike. ![file] , 
 Dear Dr. Singh, it is always greatly appreciated if authors reply to concerns, and I am very grateful that you were able to pull up the original blots to address Actinopolyspora Biskrensis concerns. However, although I agree these look like two different films, the spacing, angles, and thickness of the bands are much more similar between these two films as one would expect if these were two blots. I am not talking about the angle of the whole blot, but the small differences in angles between each band, and that of the minor M band at the far right. Do you have an explanation for these unexpected similarities? , 
 Dear Dr. Bik, Thank you very much for showing interest in our work. I totally agree with you that there are similarities between the two blots. There may be a number of reasons for the same. I will try to list a few. I have already mentioned that these are the blots of two different proteins whose expression does not change much during different treatments (control proteins). The blots have been derived from same protein samples. The two proteins probed are of similar sizes (38 kDa for p38 and 37kDa for GAPDH). For saving the antibodies and the money, we have a practice of using the primary and the secondary antibodies alteast twice for the control blots. Nevertheless, I will try to bring about the differences in the two blots in terms of band angle and the band intensity (specially the M band). In the image (A), I have tried to bring out the differences in the band angle in the two gels which is different. In the image (B), I have shown the band intensities of the two gels and normalized fold changes wrt to the lane 1 (macrophage cnt). The fold change is similar but not exactly the same for the remaining lanes again emphasizing that the levels of p38 and GAPDH do not change in different treatments. However, the normalized intensity of the non-specific band is different in the two gels. I hope this answers your query. Thank you ![file] ,