Human Transcription Release Factor 2 Dissociates RNA Polymerases I and II Stalled at a Cyclobutane Thymine Dimer

RNA polymerase II stalled at a lesion in the transcribed strand is thought to constitute a signal for transcription-coupled repair. Transcription factors that act on RNA polymerase in elongation mode potentially influence this mode of repair. Previously, it was shown that transcription elongation factors TFIIS and Cockayne's syndrome complementation group B protein did not disrupt the ternary complex of RNA polymerase II stalled at a thymine cyclobutane dimer, nor did they enable RNA polymerase II to bypass the dimer. Here we investigated the effect of the transcription factor 2 on RNA polymerase II and RNA polymerase I stalled at thymine dimers. Transcription factor 2 is known to release transcripts from RNA polymerase II early elongation complex generated by pulse-transcription. We found that factor 2 (which is also called release factor) disrupts the ternary complex of RNA polymerase II at a thymine dimer and surprisingly exerts the same effect on RNA polymerase I. These findings show that in mammalian cells a RNA polymerase I or RNA polymerase II transcript truncated by a lesion in the template strand may be discarded unless repair is accomplished rapidly by a mechanism that does not displace stalled RNA polymerases

Automatic quantification of mammary glands on non-contrast X-ray CT by using a novel segmentation approach

ABSTRACT This paper describes a brand new automatic segmentation method for quantifying volume and density of mammary gland regions on non-contrast CT images. The proposed method uses two processing steps: (1) breast region localization, and (2) breast region decomposition to accomplish a robust mammary gland segmentation task on CT images. The first step detects two minimum bounding boxes of left and right breast regions, respectively, based on a machine-learning approach that adapts to a large variance of the breast appearances on different age levels. The second step divides the whole breast region in each side into mammary gland, fat tissue, and other regions by using spectral clustering technique that focuses on intra-region similarities of each patient and aims to overcome the image variance caused by different scan-parameters. The whole approach is designed as a simple structure with very minimum number of parameters to gain a superior robustness and computational efficiency for real clinical setting. We applied this approach to a dataset of 300 CT scans, which are sampled with the equal number from 30 to 50 years-old-women. Comparing to human annotations, the proposed approach can measure volume and quantify distributions of the CT numbers of mammary gland regions successfully. The experimental results demonstrated that the proposed approach achieves results consistent with manual annotations. Through our proposed framework, an efficient and effective low cost clinical screening scheme may be easily implemented to predict breast cancer risk, especially on those already acquired scans

The SWI/SNF Chromatin-Remodeling Factor Stimulates Repair by Human Excision Nuclease in the Mononucleosome Core Particle

To investigate the role of chromatin remodeling in nucleotide excision repair, we prepared mononucleosomes with a 200-bp duplex containing an acetylaminofluorene-guanine (AAF-G) adduct at a single site. DNase I footprinting revealed a well-phased nucleosome structure with the AAF-G adduct near the center of twofold symmetry of the nucleosome core. This mononucleosome substrate was used to examine the effect of the SWI/SNF remodeling complex on the activity of human excision nuclease reconstituted from six purified excision repair factors. We found that the three repair factors implicated in damage recognition, RPA, XPA, and XPC, stimulate the remodeling activity of SWI/SNF, which in turn stimulates the removal of the AAF-G adduct from the nucleosome core by the excision nuclease. This is the first demonstration of the stimulation of nucleotide excision repair of a lesion in the nucleosome core by a chromatin-remodeling factor and contrasts with the ACF remodeling factor, which stimulates the removal of lesions from internucleosomal linker regions but not from the nucleosome core

Effect of Damage Type on Stimulation of Human Excision Nuclease by SWI/SNF Chromatin Remodeling Factor

To investigate the repair of different types of DNA lesions in chromatin, we prepared mononucleosomes containing an acetylaminofluorene-guanine adduct (AAF-G), a (6-4) photoproduct, or a cyclobutane pyrimidine dimer (CPD) and measured the repair of these lesions by reconstituted 6-factor human excision nuclease. We find that incorporation into nucleosomes inhibits the repair of CPD more severely than repair of the AAF-G adduct and the (6-4) photoproduct. Equally important, we find that SWI/SNF stimulates the removal of AAF-G and (6-4) photoproduct but not of CPD from nucleosomal DNA. These results shed new light on the low rate of repair of CPDs in human cells in vivo

DNA Damage in the Nucleosome Core Is Refractory to Repair by Human Excision Nuclease

To investigate the effect of nucleosomes on nucleotide excision repair in humans, we prepared a mononucleosome containing a (6-4) photoproduct in the nucleosome core and examined its repair with the reconstituted human excision nuclease system and with cell extracts. Nucleosomal DNA is repaired at a rate of about 10% of that for naked DNA in both systems. These results are in agreement with in vivo data showing a considerably slower rate of repair of overall genomic DNA relative to that for transcriptionally active DNA. Furthermore, our results indicate that the first-order packing of DNA in nucleosomes is a primary determinant of slow repair of DNA in chromatin