Defining genome maintenance pathways using functional genomic approaches

Abstract

Genome maintenance activities including DNA repair, cell division cycle control, and checkpoint signaling pathways preserve genome integrity and prevent disease. Defects in these pathways cause birth defects, neurodegeneration, premature aging, and cancer. Recent technical advances in functional genomic approaches such as expression profiling, proteomics, and RNA interference (RNAi) technologies have rapidly expanded our knowledge of the proteins that work in these pathways. In this review, we examine the use of these high-throughput methodologies in higher eukaryotic organisms for the interrogation of genome maintenance activities.

Figure 1. Genome maintenance requires the coordination of multiple cellular activities

(A) Multiple DNA repair pathways operate to remove DNA lesions caused by endogenous and exogenous genotoxic agents. (B) DNA repair and metabolism occurs in the context of chromatin. Chromatin modifications regulate protein access to the DNA as well as signaling responses to DNA damage. (C) DNA replication must faithfully duplicate the DNA and chromatin structure once and only once per cell division cycle. (D) Proper spindle assembly and chromosome segregation during mitosis ensures each daughter cell receives a complete copy of the genome. (E) Cell cycle checkpoints monitor DNA damage, replication, and mitosis.

Figure 2. Proteomic approaches are useful to identify genome maintenance proteins

(A) Protein microarrays can be used to identify post-translational modifications (A1) and protein-protein interactions (A2). For example, Merbl et al., identified substrates of the APC ubiquitin conjugating enzyme using protein microarrays (Merbl and Kirschner, 2009). (A2) Protein interactions can be visualized on protein microarrays using fluorescently tagged proteins as the probe. (B) Yeast 2 hybrid screens detect interactions between bait proteins and prey proteins. Although useful to interrogate pairs of proteins (B1), these screens are easily easily expanded to screen cDNA libraries to generate interactome maps (B2). (C) Affinity purification coupled to mass spectrometry is useful to interrogate protein complexes purified from their native cellular context or as depicted, to identify differential post-translational modifications following genotoxic stress. (D) Analysis of protein localization is a good indicator of function within genome maintenance pathways. (D1) Fluorescence microscopy to examine subcellular localization can identify proteins that change localization in response to genotoxic drugs like hydroxyurea (HU). (D2) Biochemical purification of cellular components coupled to mass spectrometry provides an alternative method of defining proteins that reside in cellular structures relevant to genome maintenance activities.

Figure 3. RNAi screens provide a genetic approach to identifying genome maintenance activities

(A) Pooled format screens facilitate drug hyper-sensitivity screening. (A1) Barcoded shRNA vector libraries are introduced into a population of cells, (A2) split into treated and untreated groups, (A3) grown for several generations to allow selection against a subset of the shRNA expressing cells; and (A4) analyzed by sequencing or microarray technology to identify differences in shRNA abundance. (B) Arrayed format RNAi libraries can be used both for (B1) hyper-sensitivity or (B2) high-content screens.