Page 90 - 2014 Printable Abstract Book
P. 90
and the concomitant roles of these factors in DSB repair will be discussed. The influence of loss of function
(SNPs or loss of expression of these factors) on carcinogenesis will be discussed.
(S2501) RNA recognition motif-containing proteins, SFPQ (PSF) and NONO (p54nrb) define a distinct,
2
1
parallel pathway of DNA double-strand break repair. William S. Dynan ; Lahcen Jaafar, PhD ; Shuyi Li,
2
2
2
2
3
2
2
MD PhD ; Zhentian Li ; Fengjue Shu ; Hairong Xiong ; Liliana Souza ; Erica Silva ; and Morgan McLemore
2
1
Institute of Molecular Medicine and Genetics, Augusta, GA ; Emory University, Atlanta, GA ; and Wuhan
3
University, Wuhan, China
Three genes, SFPQ, NONO, and PSPC1, encode a small family of tandem RRM domain-containing
proteins with diverse functions in RNA processing, transcription, and DNA repair. Previously, we showed
that an SFPQ•NONO complex promotes nonhomologous end joining (NHEJ) in vitro. We have now
developed a reconstituted DNA end joining system, based entirely on recombinant factors, and we have
used this to demonstrate that the SFPQ•NONO complex substitutes for XLF (a canonical NHEJ factor) and
vice versa. The XLF and SFPQ•NONO sub-pathways produce a similar distribution of concatemerized
products in vitro, although there are some differences in DNA substrate preference. 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. To further investigate the in vivo
function of SFPQ•NONO-directed DNA repair, we generated knockout mice lacking the murine NONO
homolog. We investigated hematopoietic stem cells (HSCs), which are known to be sensitive to DNA repair
gene mutations. In the knockout mice, the proportion of actively cycling HSCs was much higher than
normal, and the levels of reactive oxygen species were also elevated. Moreover, the HSCs from knockout
mice were at a severe disadvantage in a competitive bone marrow transplant. We also investigated the
testis, based on observations that male, but not female, knockout mice were smaller than their wild-type
counterparts. Testes of knockout mice failed to develop normally in a critical period between postnatal
days 18 and 24. They were also radiosensitive. The testes of wild-type mice showed no immediate effects
following exposure to 4 Gy of γ-ray. By contrast, in the knockout mice, elevated levels of apoptotic cells
were evident within seminiferous tubules at 24 h following irradiation, and there was a nearly complete
absence of germ cells at 48 h following irradiation. The co-occurrence of hematopoietic and germ cell
defects is reminiscent of other DNA repair gene mutations, including Fanconi anemia. Together, results
indicate that the SFPQ•NONO complex promotes a sub-pathway of double strand break repair with
distinct biological functions. Although the presence of RRM domains in SFPQ and NONO suggests that
pathway activity is modulated by RNA, the pathway is able to function in vitro in the absence of RNA, and
thus the exact role of RNA binding remains to be defined.
(S2502) Investigating the role of Xrn2 in the DNA damage response and genomic instability.
Julio Morales, UT Southwestern, Dallas, TX
The role of transcriptional by-products as a source of genomic instability and the DNA damage
response is becoming more apparent 1, 2 . Several studies have found that factors classically viewed in
context of RNA metabolism are also intimately involved in the DNA damage response (DDR), with the best
3-8
examples being factors involved in transcription termination . Transcription termination is a process that
9
involved several proteins . Several transcription termination factors have been shown to have direct roles
88 | P a g e
(SNPs or loss of expression of these factors) on carcinogenesis will be discussed.
(S2501) RNA recognition motif-containing proteins, SFPQ (PSF) and NONO (p54nrb) define a distinct,
2
1
parallel pathway of DNA double-strand break repair. William S. Dynan ; Lahcen Jaafar, PhD ; Shuyi Li,
2
2
2
2
3
2
2
MD PhD ; Zhentian Li ; Fengjue Shu ; Hairong Xiong ; Liliana Souza ; Erica Silva ; and Morgan McLemore
2
1
Institute of Molecular Medicine and Genetics, Augusta, GA ; Emory University, Atlanta, GA ; and Wuhan
3
University, Wuhan, China
Three genes, SFPQ, NONO, and PSPC1, encode a small family of tandem RRM domain-containing
proteins with diverse functions in RNA processing, transcription, and DNA repair. Previously, we showed
that an SFPQ•NONO complex promotes nonhomologous end joining (NHEJ) in vitro. We have now
developed a reconstituted DNA end joining system, based entirely on recombinant factors, and we have
used this to demonstrate that the SFPQ•NONO complex substitutes for XLF (a canonical NHEJ factor) and
vice versa. The XLF and SFPQ•NONO sub-pathways produce a similar distribution of concatemerized
products in vitro, although there are some differences in DNA substrate preference. 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. To further investigate the in vivo
function of SFPQ•NONO-directed DNA repair, we generated knockout mice lacking the murine NONO
homolog. We investigated hematopoietic stem cells (HSCs), which are known to be sensitive to DNA repair
gene mutations. In the knockout mice, the proportion of actively cycling HSCs was much higher than
normal, and the levels of reactive oxygen species were also elevated. Moreover, the HSCs from knockout
mice were at a severe disadvantage in a competitive bone marrow transplant. We also investigated the
testis, based on observations that male, but not female, knockout mice were smaller than their wild-type
counterparts. Testes of knockout mice failed to develop normally in a critical period between postnatal
days 18 and 24. They were also radiosensitive. The testes of wild-type mice showed no immediate effects
following exposure to 4 Gy of γ-ray. By contrast, in the knockout mice, elevated levels of apoptotic cells
were evident within seminiferous tubules at 24 h following irradiation, and there was a nearly complete
absence of germ cells at 48 h following irradiation. The co-occurrence of hematopoietic and germ cell
defects is reminiscent of other DNA repair gene mutations, including Fanconi anemia. Together, results
indicate that the SFPQ•NONO complex promotes a sub-pathway of double strand break repair with
distinct biological functions. Although the presence of RRM domains in SFPQ and NONO suggests that
pathway activity is modulated by RNA, the pathway is able to function in vitro in the absence of RNA, and
thus the exact role of RNA binding remains to be defined.
(S2502) Investigating the role of Xrn2 in the DNA damage response and genomic instability.
Julio Morales, UT Southwestern, Dallas, TX
The role of transcriptional by-products as a source of genomic instability and the DNA damage
response is becoming more apparent 1, 2 . Several studies have found that factors classically viewed in
context of RNA metabolism are also intimately involved in the DNA damage response (DDR), with the best
3-8
examples being factors involved in transcription termination . Transcription termination is a process that
9
involved several proteins . Several transcription termination factors have been shown to have direct roles
88 | P a g e
