p53 promotes the fidelity of DNA end-joining activity by, in part, enhancing the expression of heterogeneous nuclear ribonucleoprotein G

Abstract

Many studies have suggested the involvement of wild-type (wt) p53 in the repair of DNA double-strand breaks (DSBs) via DNA end-joining (EJ) process. To investigate this possibility, we compared the capacity and fidelity of DNA EJ in RKO cells containing wt p53 and RKO cells containing no p53 (RKO cells with p53 knockdown). The p53 knockdown cells showed lower fidelity of DNA EJ compared to the control RKO cells. The DNA end-protection assay revealed the association of a protein complex including heterogeneous nuclear ribonucleoprotein G (hnRNP G) with the DNA ends in RKO cells containing wt p53, but not with the DNA ends in RKO cells with p53 knockdown. Depletion of endogenous hnRNP G notably diminished the fidelity of EJ in RKO cells expressing wt p53. Moreover, an ectopic expression of hnRNP G significantly enhanced the fidelity of DNA EJ and the protection of DNA ends in human cancer cells lacking hnRNP G protein or containing mutant hnRNP G. Finally, using recombinant hnRNP G proteins, we demonstrated the hnRNP G protein is able to bind to and protect DNA ends from degradation of nucleases. Our results suggest that wt p53 modulates DNA DSB repair by, in part, inducing hnRNP G, and the ability of hnRNP G to bind and protect DNA ends may contribute its ability to promote the fidelity of DNA EJ.

hnRNP G elicits tumor-suppressive activity in part by upregulating the expression of Txnip

“…Our previous study showed that the HEp-2 cells lack endogenous hnRNP G expression and are a proper cell line to test effect of exogenous hnRNP G on various biochemical processes [8,9]. Indeed, we demonstrated that wt hnRNP G elicited tumor suppressive activity, and its overexpression led to marked induction of Txnip mRNA in HEp-2 cells [8].…”

“…In fact, there is no report describing the presence of a DNA binding domain in hnRNP G protein. Apart from its function in RNA splicing, the other physiological roles of hnRNP G have yet to be fully understood and may include tumor suppression and DNA repair mechanisms [8,9]. Recent studies have shown that several hnRNP family proteins, such as D0B, E2BP, and K, are able to bind to the promoters of various cellular genes [10][11][12].…”

“…Recent studies have shown that several hnRNP family proteins, such as D0B, E2BP, and K, are able to bind to the promoters of various cellular genes [10][11][12]. It is therefore interesting to note the DNA end binding ability of recombinant hnRNP G [9]. Moreover, a recent study showed that hnRNP G regulates the activity of the sterol regulatory element-binding protein-1c promoter [7].…”

“…The PCR fragments were then cloned into the pLNCX2 retrovirus expression plasmid (Clontech) digested with XhoI and HindIII. The resulting construct, pLNCX2-Txnip, was transfected into PA317 packaging cell line to harvest the viral vectors as described previously [8,9]. Cells were infected with the viral vetors and selected with G418 (100 μg/ml).…”

Expression of RBMX in the Light-Induced Damage of Rat Retina In Vivo

“…RNA-binding motif protein, X-linked (RBMX) is a heterogeneous nuclear ribonucleoprotein (Shin et al 2008a) and is made of 391 amino acid residues containing the RNA binding domain at the amino terminus of the protein (Shin et al 2007). This architectural feature exposits the physical association of RBMX with RNA, which may be required in RNA processing and metabolism (Shin et al 2006(Shin et al , 2007.…”