Role of SIRT1 and SIRT6 for the preservation of genetic stability
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Abstract
Genome stability is very important for all living organisms and is constantly under threat because of the exposure to different environmental and endogenous DNA damaging agents. Genome instability, for example in consequence of defective DNA repair, is an important risk factor for the initiation of carcinogenesis. Aim of this study was to investigate the influence of SIRT1 and SIRT6 on genome stability. Sirtuins are NAD+-dependent deacetylases of histones and other proteins that could influence base excision repair (BER) by modulating chromatin accessibility, by modifying enzymes involved in DNA repair or by affecting metabolism and the production of reactive oxygen species (ROS) that will increase DNA damage load.
For investigation of these hypotheses, SIRT1 overexpressing and deficient cells were used, as well as Sirt6-/- knock-out and Sirt6-/-/Ogg1-/- double knock-out. The micronucleus assay was used as indicator for genome instability. DNA damage and repair were measured by an alkaline elution assay. A DNA relaxation assay was used for the analyses of APE1 activity.
In this study, a mechanism for micronuclei generation from SSBs as a result of impaired base excision repair pathway due to SIRT1-modulated APE1 activity was proposed. More SSBs in SIRT1 deficient cells lead to higher number of micronuclei, as result of increased APE1 cleavage activity and genomic instability. Reduced APE1-endonuclease activity in SIRT1 overexpressing cells results in less SSBs generation and lower number of micronuclei, suggesting a protective role of SIRT1 on genome stability. Interestingly, the activity of SIRT1 was found to be stimulated by oxidative stress. Thus the APE1 activity in cell extracts was reduced after treatment with bromate in normal, but not in SIRT1 deficient cells, while SIRT1-overexpressing cells exhibit low APE1 activity, independent of oxidative stress. An unexpected accumulation of AP sites and SSBs in MMS-treated SIRT1-deficient cells suggests an additional influence of SIRT1 on the activity of methyl purine glycosylase (MPG), another repair enzyme.
In summary, the results demonstrate that SIRT1 plays a previously unknown role in the preservation of the genetic stability of cells. By its influence on the processing of AP sites (via a regulation of APE1 activity) it prevents an unbalanced base excision repair, in particular an accumulation of SSBs as repair intermediates, which would cause chromosome breaks and micronuclei formation.
Many questions regarding the relevance of SIRT6 for genetic stability remain unanswered due to the low number and early death of SIRT6 deficient animals that could be generated. However, results in this study clearly demonstrate that SIRT6 has no influence on the resealing of SSBs. An unexpected protective role of SIRT6 deficiency on genome stability, which was only observed in combination with OGG1 deficiency, raises new questions about a mechanistic interaction / interplay of these two proteins. Further investigations (and more animals) are needed for any conclusions about the role of SIRT6 on genome stability and the proteins involved.