Literature Review: Our Current Understanding of the Mechanism Underlying Micronuclei-Mediated Mutagenesis in Cancer

Abstract

Purpose: The purpose of the experimental research and overall review presented within this paper is to gain a comprehensive understanding of where the field of study on micronuclei currently stands, with a specific focus on the mechanism by which micronuclei mediate DNA damage and actively contribute to mutagenesis. The purpose of the review is to establish what is known so as to point to future directions for further mechanistic insight and potential targetable areas for therapeutic benefit. Methods: A comprehensive literature search of modern and more historic literature was conducted for inclusivity and reported upon in detail in writing throughout this paper. As for the actual preliminary experimental data and working hypothesis presented in the section entitled “Working Hypothesis: Replication catastrophe in the setting of limited supplies”, please see the associated section for more details. In short, our working hypothesis regarding the mechanism of micronuclei-mediated DNA damage stemmed from the fact that micronuclei exhibit abnormal DNA replication and localization of key replication factors including Replication Factor A (RPA) and subunits of DNA helicase. As a result, we hypothesized that replicating micronuclei may be subject to increased replication stress leading to long stretches of single-stranded DNA (ssDNA), thus providing a substrate for cytoplasmic nucleases upon rupture. In order to test the hypothesis, we assessed the extent of ssDNA present in micronuclei in S phase using a native BrdU assay using U2OS and RPE1 cell lines using a well-established method for inducing micronucleation using a microtubule poison, nocodazole. Results: We observed fewer BrdU foci in intact replicating micronuclei as compared to primary nuclei, pointing to less efficient replication. Even upon induction of replication stress in the setting of ATR inhibition that leads to massive dormant origin activation in primary nuclei and subsequent replication catastrophe, we observed minimal increase in BrdU foci in intact micronuclei, suggesting that micronuclei may have few functional origins of replication. In contrast, micronuclei with disrupted nuclear envelopes (NEs) exhibited dense clusters of BrdU foci. Since these micronuclei are not expected to undergo further replication upon loss of membrane integrity, we postulate that the observed foci likely represent the sites of DNA double-strand breaks (DSBs) that form upon NE rupture in the setting of DNA replication. The results of the literature search that help to contextualize these findings within the micronuclei research field is included throughout the text below. Conclusions: The initial experimental results point to a defect in origin licensing as a cause of replication delay in micronuclei, thus providing a link between aberrant replication, replication stress, and ensuing mutagenesis. Further support for why and how micronuclei may be subject to defective replication and inadequate materials for origin licensing leading to replication catastrophe with ensuing DSB formation and DNA damage is included throughout the text of this paper. Because micronuclei can contribute a large number of mutations within a single cycle, they may plan an important role during the process of tumorigenesis. Understanding the process by which micronuclei lead to accumulation of DNA mutations and tumor formation will accelerate the development of effective preventative and therapeutic strategies for patients with cancer.