NHEJ, Ku, IP6, DNApäädyt, DNA.PK(cs)

PubMed antaa kaksi vastausta:

Synthesis of biotinylated inositol hexakisphosphate to study DNA double-strand break repair and affinity capture of IP6-binding proteins.

Jiao C, Summerlin M, Bruzik KS, Hanakahi L.

Biochemistry. 2015 Sep 23. [Epub ahead of print]

PMID:

26397942

Biochemistry. 2015 Sep 23. [Epub ahead of print]

Synthesis of biotinylated inositol hexakisphosphate to study DNA double-strand break repair and affinity capture of IP6-binding proteins.

Jiao C, Summerlin M, Bruzik KS, Hanakahi L.

Abstract

Inositol hexakisphosphate (IP6) is a soluble inositol polyphosphate, which is abundant in mammalian cells. Despite participation of IP6 in critical cellular functions, few IP6-binding proteins have been characterized.

We report on synthesis, characterization and application of biotin-labeled IP6 (IP6-biotin), which has biotin attached at the 2-position of myo-inositol ring via an aminohexyl linker. Like natural IP6, IP6-biotin stimulated DNA ligation by non-homologous end joining (NHEJ) in vitro.

 The Ku protein is a required NHEJ factor that has been shown to bind IP6. We found that IP6-biotin could affinity-capture Ku and other required NHEJ factors from human cell extracts, including the DNA-dependent protein kinase catalytic subunit (DNA-PKcs), XRCC4 and XLF.

 Direct binding studies with recombinant proteins show that Ku is the only NHEJ factor with affinity for IP6-biotin. DNA-PKcs, XLF and the XRCC4:Ligase IV complex interact with Ku in cell extracts and likely interact indirectly with IP6-biotin.

IP6-biotin was used to tether streptavidin to Ku, which inhibited NHEJ in vitro. These proof-of-concept experiments suggest that molecules like IP6-biotin might be used to molecularly target biologically important proteins that bind IP6. IP6-biotin affinity capture experiments show that numerous proteins specifically bind IP6-biotin, including casein kinase 2 (CK2), which is known to bind IP6, and nucleolin

. Protein binding to IP6-biotin is selective, as IP3, IP4 and IP5 did not compete for binding of proteins to IP6-biotin. Our results document IP6-biotin as a useful tool to investigate the role of IP6 in biological systems.

PMID:

26397942

[PubMed - as supplied by publisher]

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Select item 118213782.

Binding of inositol hexakisphosphate (IP6) to Ku but not to DNA-PKcs.

Ma Y, Lieber MR.

J Biol Chem. 2002 Mar 29;277(13):10756-9. Epub 2002 Jan 30.

PMID:

11821378

Free Article

J Biol Chem. 2002 Mar 29;277(13):10756-9. Epub 2002 Jan 30.

Binding of inositol hexakisphosphate (IP6) to Ku but not to DNA-PKcs.

Ma Y¹, Lieber MR.

Abstract (2002)

The nonhomologous DNA end joining (NHEJ) pathway is responsible for repairing a major fraction of double strand DNA breaks in somatic cells of all multicellular eukaryotes. As an indispensable protein in the NHEJ pathway, Ku has been hypothesized to be the first protein to bind at the DNA ends generated at a double strand break being repaired by this pathway. When bound to a DNA end, Ku improves the affinity of another DNA end-binding protein, DNA-PK(cs), to that end. The Ku.DNA-PK(cs) complex is often termed the DNA-PK holoenzyme. It was recently shown that myo-inositol hexakisphosphate (IP(6)) stimulates the joining of complementary DNA ends in a cell free system. Moreover, the binding data suggested that IP(6) bound to DNA-PK(cs) (not to Ku). Here we clearly show that, in fact, IP(6) associates not with DNA-PK(cs), but rather with Ku. Furthermore, the binding of DNA ends and IP(6) to Ku are independent of each other. The possible relationship between inositol phosphate metabolism and DNA repair is discussed in light of these findings.

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DNA:n korjauksesta uutta 2015 . Fokus: NHEJ E3 ub. ligaasi APC

Haen  lähtökohtana Per Arne Aasin nimi ja  DSB , DNA:n korjausmekanismit.
Ne tiedot voat 2004 ajalta. Nyt luin siitä että fytiini on mukana NHEJ:ssä Joten otan nämä asiat esiin.

Löydän tämän: http://www.sciencedirect.com/science/article/pii/S1568786415000737

Cell cycle regulation of human DNA repair and chromatin remodeling genes

• Robin Mjelle^a,
• Siv Anita Hegre^a,
• Per Arne Aas^a,
• Geir Slupphaug^a,
• Finn Drabløs^a,
• Pål Sætrom^{a, b, ,} ,
• Hans E. Krokan^{a, ,}

Under a Creative Commons license

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6.10.2015 Tähän otan sitaatin siitä NHEJ- asiasta, jossa fytiini(IP6) jota solussa pitäisi olla runsaasti,, rekrytoi Ku suoraan, ja epäsuorasti  sitten muuta kuten alla mainittua molekyyliä XRCC6.

3.10. Genes encoding proteins for non-homologous end joining (NHEJ) – a regulatory role for PTTG/securin

Generally, genes centrally involved in NHEJ were found not to be cell cycle regulated (Fig. 8). However, PTTG1, encoding the 202 amino acids protein PTTG1 (also called securin), consistently had the highest transcript level at the M/G1-transition in all data sets and then declines rapidly during the G1 phase. PTTG1 is a multifunctional and rather intriguing protein. It binds directly to Ku70 (also called XRCC6) and functions as a negative regulator of NHEJ [73] and [74]. Thus, PTTG1 may inhibit illegitimate NHEJ until completion of replication.

 It was reported that PTTG1 is ubiquitinated at metaphase-anaphase transition by the E3 ligase anaphase-promoting complex (APC). It is then normally rapidly degraded in proteasomes (reviewed in [75]), thus alleviating the NHEJ-inhibition and reestablishing the capacity for NHEJ during G1 phase.

Importantly, PTTG1 also controls sister chromatid separation during mitosis by binding to and inhibiting separase, which when active cleaves cohesin that holds sister chromatids together [76]. Normal levels of PTTG1 may prevent premature sister chromatid separation, thus facilitating DNA repair by HRR during the S phase and G2.

 However, overexpression of PTTG1 correlates strongly with aneuploidy in breast cancer [77], possibly due to an inability of sister chromatids to separate. Overexpression of PTTG1 is associated with a wide range of malignancies, e.g. hematopoietic malignancies [78], colon cancer [79] and breast cancer [80].

 Thus, while PTTG1 normally regulates several processes important to genomic stability, overexpression appears to promote genomic instability, stimulate proliferation and contribute to carcinogenesis. PTTG1 also functions as a transcriptional regulator of several genes involved in tumorigenesis [81].

 We conclude that normally the transcript level of PTTG1 is upregulated at the M/G1-transition, but subsequently ubiquitinylated and degraded in proteasomes at metaphase-anaphase transition, allowing chromatid separation.

Overexpression of PTTG1 apparently results in inability of sister chromatids to separate with increased risk of aneuploidy in cancer cells [82] and [83].