Page 121 - 2014 Printable Abstract Book
P. 121
mouse ES cells lacking Rad9. Thus, we demonstrate that Rad9 regulates base excision repair by controlling
Neil1 protein levels, albeit by somewhat different mechanisms in human versus mouse cells.
(PS1-33) SFPQ•NONO, a RNA recognition motif-containing DNA double-strand break repair factor,
participates in a parallel, XLF-independent, nonhomologous end joining pathway. Lahcen Jaafar and
William S. Dynan, Emory University, Atlanta, GA
Non-homologous end joining (NHEJ) is the default pathway for DNA double-strand break repair in
mammalian cells. The classical NHEJ pathway begins with binding of the Ku70•Ku80 complex and DNA-
PKcs to broken DNA ends, which is followed by recruitment of a DNA Ligase IV•XRCC4 complex and
XLF/Cernunnos. Recent work has demonstrated, however, that in addition to the classical NHEJ pathway,
there are parallel end joining pathways that differ in their factor requirements. Here, we characterize one
of these pathways, which requires a heterodimeric complex of two RNA recognition motif-containing
proteins, SFPQ and NONO. In addition to participation in DNA repair, these proteins have established roles
in transcription and RNA processing, and perhaps provide a link between DNA repair and RNA metabolism.
We developed a reconstituted end-joining system containing recombinant SFPQ•NONO, DNA Ligase
IV•XRCC4, and Ku70•Ku80 complexes. The first two of these were sufficient for end-joining in vitro, in a
reaction that is further stimulated by the Ku70•Ku80 complex. Surprisingly, XLF/Cernunnos, which is
required for classical NHEJ, was not required for end-joining in the presence of SFPQ•NONO, indicating
that the latter participates in a separate, parallel end-joining pathway. The XLF and SFPQ•NONO pathways
produce a similar distribution of concatemerized products in vitro, with no preference for head-head,
head to tail, or tail-to-tail plasmid joining. The XLF pathway was somewhat more efficient for joining of
small DNA fragments (<700 bp). The finding that XLF is required for only one of several pathways of NHEJ
provides an explanation for long-standing observations that XLF is less abundant than other core NHEJ
factors. SFPQ and NONO appear to be present only in metazoan organisms. More work is in progress to
determine if the SFPQ•NONO and XLF pathways are preferentially used in different cell types, or if they
specialize in repair of different types of DNA breaks.
(PS1-34) The role of RNF126 in the response to DNA double strand breaks via regulation of E2F1. Junran
1
1
1
1
3
Zhang ; Ying Wang ; Ou Deng ; Zhihui Feng ; Zhanwen Du ; Xiahui Xiong ; Daniel Li ; Ceshi Chen ; and
1
1
2
2
1
4
Zhefu Ma, Case Western Reserve University, Cleveland, OH ; Duke University, Durham, NC ; Key
Laboratory of Animal Models and Human Disease Mechanisms of Chinese Academy of Sciences, Kunming,
China ; and The First Affiliated Hospital of Sun Yat-sen University, Guangzhou, China
4
3
Defects in ubiquitin E3 ligases are implicated in the pathogenesis of several human diseases,
including cancer. However, the functional study about the existing E3 ligase is limited. It has been
demonstrated that E3 ligase RNF126 promotes the growth of breast cancer cells by targeting p21 for
ubiquitination dependent degradation. Other than that, the specific functions of RNF126 remain largely
unknown. DNA double-strand breaks (DSBs) are the most cytotoxic forms of DNA damage. Three pathways
are involved in the repair of DNA DSBs: homologous recombination (HR), classical non-homologous end
joining (C-NHEJ) and alternative NHEJ (Alt-NHEJ). Defect in DSBs repair usually leads to the sensitivity to
radiation therapy and chemotherapy. In this study, we demonstrate that RNF126 is important for HR
pathway. In support of this observation, we found that RNF126 promotes the expression of HR protein
119 | P a g e
Neil1 protein levels, albeit by somewhat different mechanisms in human versus mouse cells.
(PS1-33) SFPQ•NONO, a RNA recognition motif-containing DNA double-strand break repair factor,
participates in a parallel, XLF-independent, nonhomologous end joining pathway. Lahcen Jaafar and
William S. Dynan, Emory University, Atlanta, GA
Non-homologous end joining (NHEJ) is the default pathway for DNA double-strand break repair in
mammalian cells. The classical NHEJ pathway begins with binding of the Ku70•Ku80 complex and DNA-
PKcs to broken DNA ends, which is followed by recruitment of a DNA Ligase IV•XRCC4 complex and
XLF/Cernunnos. Recent work has demonstrated, however, that in addition to the classical NHEJ pathway,
there are parallel end joining pathways that differ in their factor requirements. Here, we characterize one
of these pathways, which requires a heterodimeric complex of two RNA recognition motif-containing
proteins, SFPQ and NONO. In addition to participation in DNA repair, these proteins have established roles
in transcription and RNA processing, and perhaps provide a link between DNA repair and RNA metabolism.
We developed a reconstituted end-joining system containing recombinant SFPQ•NONO, DNA Ligase
IV•XRCC4, and Ku70•Ku80 complexes. The first two of these were sufficient for end-joining in vitro, in a
reaction that is further stimulated by the Ku70•Ku80 complex. Surprisingly, XLF/Cernunnos, which is
required for classical NHEJ, was not required for end-joining in the presence of SFPQ•NONO, indicating
that the latter participates in a separate, parallel end-joining pathway. The XLF and SFPQ•NONO pathways
produce a similar distribution of concatemerized products in vitro, with no preference for head-head,
head to tail, or tail-to-tail plasmid joining. The XLF pathway was somewhat more efficient for joining of
small DNA fragments (<700 bp). The finding that XLF is required for only one of several pathways of NHEJ
provides an explanation for long-standing observations that XLF is less abundant than other core NHEJ
factors. SFPQ and NONO appear to be present only in metazoan organisms. More work is in progress to
determine if the SFPQ•NONO and XLF pathways are preferentially used in different cell types, or if they
specialize in repair of different types of DNA breaks.
(PS1-34) The role of RNF126 in the response to DNA double strand breaks via regulation of E2F1. Junran
1
1
1
1
3
Zhang ; Ying Wang ; Ou Deng ; Zhihui Feng ; Zhanwen Du ; Xiahui Xiong ; Daniel Li ; Ceshi Chen ; and
1
1
2
2
1
4
Zhefu Ma, Case Western Reserve University, Cleveland, OH ; Duke University, Durham, NC ; Key
Laboratory of Animal Models and Human Disease Mechanisms of Chinese Academy of Sciences, Kunming,
China ; and The First Affiliated Hospital of Sun Yat-sen University, Guangzhou, China
4
3
Defects in ubiquitin E3 ligases are implicated in the pathogenesis of several human diseases,
including cancer. However, the functional study about the existing E3 ligase is limited. It has been
demonstrated that E3 ligase RNF126 promotes the growth of breast cancer cells by targeting p21 for
ubiquitination dependent degradation. Other than that, the specific functions of RNF126 remain largely
unknown. DNA double-strand breaks (DSBs) are the most cytotoxic forms of DNA damage. Three pathways
are involved in the repair of DNA DSBs: homologous recombination (HR), classical non-homologous end
joining (C-NHEJ) and alternative NHEJ (Alt-NHEJ). Defect in DSBs repair usually leads to the sensitivity to
radiation therapy and chemotherapy. In this study, we demonstrate that RNF126 is important for HR
pathway. In support of this observation, we found that RNF126 promotes the expression of HR protein
119 | P a g e
