Korean J Physiol Pharmacol.  2009 Oct;13(5):343-348. 10.4196/kjpp.2009.13.5.343.

Ser1778 of 53BP1 Plays a Role in DNA Double-strand Break Repairs

• Lee JH
• Cheong HM
• Kang MY
• Kim SY
• Kang Y

• KMID: 1982566
• DOI: http://doi.org/10.4196/kjpp.2009.13.5.343

Abstract

53BP1 is an important genome stability regulator, which protects cells against double-strand breaks. Following DNA damage, 53BP1 is rapidly recruited to sites of DNA breakage, along with other DNA damage response proteins, including gamma-H2AX, MDC1, and BRCA1. The recruitment of 53BP1 requires a tandem Tudor fold which associates with methylated histones H3 and H4. It has already been determined that the majority of DNA damage response proteins are phosphorylated by ATM and/or ATR after DNA damage, and then recruited to the break sites. 53BP1 is also phosphorylated at several sites, like other proteins after DNA damage, but this phosphorylation is not critically relevant to recruitment or repair processes. In this study, we evaluated the functions of phosphor-53BP1 and the role of the BRCT domain of 53BP1 in DNA repair. From our data, we were able to detect differences in the phosphorylation patterns in Ser25 and Ser1778 of 53BP1 after neocarzinostatin-induced DNA damage. Furthermore, the foci formation patterns in both phosphorylation sites of 53BP1 also evidenced sizeable differences following DNA damage. From our results, we concluded that each phosphoryaltion site of 53BP1 performs different roles, and Ser1778 is more important than Ser25 in the process of DNA repair.

Keyword

53BP1; Phosphorylation; Nuclear foci; DSBs; DNA repair

MeSH Terms

DNA
DNA Damage
DNA Repair
Genomic Instability
Histones
Phosphorylation
Proteins
DNA
Histones
Proteins

Figure

• 

  Fig. 1. Schematic diagram of 53BP1. Human 53BP1 is composed of 1,972 amino acids and contains several noteworthy structural features. 53BP1 possesses a number of clustered PIK phosphorylation sites (S/T-Q motif) and two repeated C-terminal BRCT domains. Additionally, 53BP1 harbors a tandem Tudor domain, a stretch rich in glycine and arginine residues.

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  Fig. 2. Phosphorylation at Ser25 and Ser1778 of 53BP1 evidenced differing patterns after NCS-induced DNA damage. (A) U2OS cells were treated with NCS (200 ng/ml) for the indicated times, and then Western blot analysis was conducted to examine the phosphorylation patterns at Ser25 and Ser1778 of 53BP1. (B) Quantitative results showed the differences in the intensity between Ser25 and Ser1778 phosphorylation after DNA damage.

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  Fig. 3. Ser 25 and Ser1778 of 53BP1 differed in nuclear foci formation. (A) U2OS cells were grown on cover slides and then treated with NCS (200 ng/ml). At 0, 1, 3, 6, 12, and 24 hours after NCS treatment, the cells were fixed and immunostained with antibodies. (B) The graph shows the average number of Ser25 and Ser1778 foci based on ∼100 nuclei per sample. The values are expressed as the means±S.D.

• 

  Fig. 4. DNA repair was finished within 24 hours after NCS treatment. (A) DNA fragmentation in U2OS cells was analyzed via a comet assay after NCS (200 ng/ml) treatment. DNA was stained with EtBr and all images were acquired at the indicated times. (B) DNA fragmentation was quantified by determining the tail moment of at least 100 comets per condition.

Reference

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