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Nuclear alpha spectrin: Critical roles in DNA interstrand cross-link repair and genomic stability.

ABSTRACT: Non-erythroid alpha spectrin (?IISp) is a structural protein which we have shown is present in the nucleus of human cells. It interacts with a number of nuclear proteins such as actin, lamin, emerin, chromatin remodeling factors, and DNA repair proteins. ?IISp's interaction with DNA repair proteins has been extensively studied. We have demonstrated that nuclear ?IISp is critical in DNA interstrand cross-link (ICL) repair in S phase, in both genomic (non-telomeric) and telomeric DNA, and in maintenance of genomic stability following ICL damage to DNA. We have proposed that ?IISp acts as a scaffold aiding to recruit repair proteins to sites of damage. This involvement of ?IISp in ICL repair and telomere maintenance after ICL damage represents new and critical functions for ?IISp. These studies have led to development of a model for the role of ?IISp in DNA ICL repair. They have been aided by examination of cells from patients with Fanconi anemia (FA), a repair-deficient genetic disorder in which a deficiency in ?IISp leads to defective ICL repair in genomic and telomeric DNA, telomere dysfunction, and chromosome instability following DNA ICL damage. We have shown that loss of ?IISp in FA cells is due to increased breakdown by the protease, µ-calpain. Importantly, we have demonstrated that this deficiency can be corrected by knockdown of µ-calpain and restoring ?IISp levels to normal. This corrects a number of the phenotypic deficiencies in FA after ICL damage. These studies suggest a new and unexplored direction for therapeutically restoring genomic stability in FA cells and for correcting numerous phenotypic deficiencies occurring after ICL damage. Developing a more in-depth understanding of the importance of the interaction of ?IISp with other nuclear proteins could significantly enhance our knowledge of the consequences of loss of ?IISp on critical nuclear processes.

SUBMITTER: Lambert MW

PROVIDER: S-EPMC4999628 | biostudies-literature | 2016 Sep

REPOSITORIES: biostudies-literature

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Nuclear alpha spectrin: Critical roles in DNA interstrand cross-link repair and genomic stability.

Lambert Muriel W MW

Experimental biology and medicine (Maywood, N.J.) 20160801 15

Non-erythroid alpha spectrin (αIISp) is a structural protein which we have shown is present in the nucleus of human cells. It interacts with a number of nuclear proteins such as actin, lamin, emerin, chromatin remodeling factors, and DNA repair proteins. αIISp's interaction with DNA repair proteins has been extensively studied. We have demonstrated that nuclear αIISp is critical in DNA interstrand cross-link (ICL) repair in S phase, in both genomic (non-telomeric) and telomeric DNA, and in maint ...[more]

PMID: 27480253

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Project description:DNA interstrand cross-links (ICLs) are products of chemotherapeutic agents and cellular metabolic processes that block both replication and transcription. If left unrepaired, ICLs are extremely toxic to cells, and ICL repair mechanisms contribute to the survival of certain chemotherapeutic resistance tumors. A critical step in ICL repair involves unhooking the cross-link. In the absence of a homologous donor sequence, the resulting gap can be filled in by a repair synthesis step involving bypass of the cross-link remnant. Here, we examine the effect of cross-link structure on the ability of unhooked DNA substrates to undergo repair synthesis in mammalian whole cell extracts. Using 32P incorporation assays, we found that repair synthesis occurs efficiently past the site of damage when a DNA substrate containing a single N4C-ethyl-N4C cross-link is incubated in HeLa or Chinese hamster ovary cell extracts. This lesion, which can base pair with deoxyguanosine, is readily bypassed by both Escherichia coli DNA polymerase I and T7 DNA polymerase in a primer extension assay. In contrast, bypass was not observed in the primer extension assay or in mammalian cell extracts when DNA substrates containing a N3T-ethyl-N3T or N1I-ethyl-N3T cross-link, whose linkers obstruct the hydrogen bond face of the bases, were used. A modified phosphorothioate sequencing method was used to analyze the ICL repair patches created in the mammalian cell extracts. In the case of the N4C-ethyl-N4C substrate, the repair patch spanned the site of the cross-link, and the lesion was bypassed in an error-free manner. However, although the N3T-ethyl-N3T and N1I-ethyl-N3T substrates were unhooked in the extracts, bypass was not detected. These and our previous results suggest that although the chemical structure of an ICL may not affect initial cross-link unhooking, it can play a significant role in subsequent processing of the cross-link. Understanding how the physical and chemical differences of ICLs affect repair may provide a better understanding of the cytotoxic and mutagenic potential of specific ICLs.

Dataset Information

Nuclear alpha spectrin: Critical roles in DNA interstrand cross-link repair and genomic stability.

Publications

Nuclear alpha spectrin: Critical roles in DNA interstrand cross-link repair and genomic stability.

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