Centromeres are chromosomal elements made up of satellite DNA, which are essential for the correct segregation of chromosomes to daughter cells in mitosis and meiosis. They are an integral part of the chromosome structure within the primary constriction and represent a structural platform to which numerous proteins are recruited for kinetochore assembly, attachment of spindle microtubules and the association of sister chromatids prior to the onset of anaphase [1]. Centromere function is ensured by the proper structural organization of centromeric proteins on specialized chromatin specified by the presence of CENP-A, the centromere-specific variant of histone H3, that serves as a key determinant of centromere identity and kinetochore assembly. While the epigenetic determinants are established, the exact contribution of centromeric DNA sequences remains unclear. The presence of repetitive DNA at centromere loci is preserved through evolution in species ranging from plants to humans implying a contribution to centromere functions [2]. In contrast, the existence of functional neocentromeres positioned on non-repetitive DNA suggests a non-essential contribution of centromere DNA that can function, once epigentically determined, in genomic regions away from satellites loci [3]. Recent evidence tries to reconcile these notions proposing that transcription of the locus is important to establish the right chromatin environment within and surrounding both endogenous centromere and neo-centromere to function proficiently [4].

The common genetic structure of the centromere comprises a modular repetition within a central core containing homogeneous repeats flanked by an outer heterochromatic domain, the pericentromere, containing less ordered repetitive elements [1]. For example, centromeres in the fission yeast Schizosaccharomyces pombe are composed of a central core region flanked by inner inverted repeats and less ordered outer repeats [5]. Along the same line, mouse core centromeres are composed by homogeneous 120 bp minor satellite repeats, flanked by less ordered 234 bp major satellite repeats [6]. Human centromeric DNA comprises a ∼171-base pair (bp) AT-rich element (alpha-satellite DNA monomer) organized in a head-to-tail tandem fashion to form higher order repeats (HORs). Megabase-sized arrays of HORs form the active centromere for each chromosome. Most of these structures contain a 17-bp centromeric motif, conserved only in primates and human, which is specifically recognized by the centromeric protein (CENP)-B [reviewed in [7], [8], [9]. CENP-B interacts and stabilizes the other centromeric proteins CENP-A and CENP-C contributing to centromere function [10]. However, because of the poor evolutionary conservation, the importance of the CENP-B box and CENP-B protein is still unclear.

Among centromeric proteins, CENP-C is another key player which is found most closely associated with the kinetochore and links the inner and outer domains. This interaction is fundamental to proper kinetochore structure allowing a connection between centromeres and microtubules during cell division. CENP-C is recruited at centromeres through the interaction with a long non-coding RNA via a triplex with centromeric RNA:DNA loops [11]. In addition to the basal centromere-specific chromatin determined by the presence of CENP-A within centromeric, alpha-satellite repeats the DNA is constitutively bound by the CCAN (Constitutive Centromere Associated Network) [12]. The CCAN is a group of 16 proteins that localize to the centromere throughout the cell cycle. Whereas in interphase the function of CCAN is mainly related to CENP-A stabilization, in mitosis it is essential for kinetochore assembly and chromosome segregation. Recent evidence on CCAN structure in vitro suggests that positioning of two CENP-A nucleosomes with a CENP-C dimer permits complete assembly of one full kinetochore [13].

The repetitive nature, specific features and structure of centromeric chromatin may negatively impact the ability of these regions to replicate accurately. During DNA replication, frequent misalignments due to the formation of hairpin-like secondary structures or the centromere-specific chromatin environment, can slow down the progression of replication forks. This may lead to changes in copy number repeat, altered structural organization or require stabilizing factors. Repetitive sequences are generally unstable and prone to recombination [14] and centromeres are hotspots for chromosome breakage in mammalian cells, even in quiescent cells [15]. Despite its gene-poor nature, centromeric DNA is actively transcribed by RNA polymerase II (RNAPII) to produce non-coding RNAs [reviewed in [16]. Centromeric transcription is critical for kinetochore assembly and for correct chromosome segregation. During transcription, transient annealing of the nascent transcript to the complementary DNA template strand generates DNA:RNA hybrid R-loops. These R-loops are a major obstacle to fork progression and cause replication stress. They are generally resolved by DNA repair proteins that promote unwinding and by exonuclease action that results in transcription termination [reviewed in [16]. Fine tuning of R-loop levels is mediated also by CENP-A, and rapid removal of CENP-A during S phase results in accumulation of R-loops and unfinished replication [17]. The presence of R-loops at centromeres during the S-phase remains, however, a contentious issue [18]. During mitosis, on the other hand, R-loops contribute to recruitment of Rad3-related kinase (ATR) at centromeres promoting faithful chromosome segregation [19] (see ATR section). The dual role of R-loops in both contributing to and hindering centromere stability is an interesting paradox that requires further exploration.

The complex structure and biology of satellite DNA sequences along with issues during replication and repair, active transcription and other peculiarities specific to these loci collectively endangers centromere integrity and renders them susceptible to chromosome rearrangements. The importance of centromeric DNA sequences is reflected in the abundance of centromere-localizing proteins that protect their integrity and reduce recombination within alpha-satellite DNA. For example, CENP-A, CENP-C, and CENP-T/W play critical roles in this protection independently of their involvement in kinetochore structure and chromosome segregation during mitosis [20]. In addition, replicating centromeric chromatin is significantly enriched for DNA repair proteins. This suggests that multiple DNA repair systems participate in maintaining the stability of centromeric DNA sequences thereby overcoming the intrinsic difficulty in maintaining sequences and functions [21].

In this review, we address how centromere-associated DNA repair and DNA damage response (DDR) proteins may participate in maintaining the structure and function of satellite DNA repeats contributing to centromere stability and possibly, function. We describe accumulating evidence regarding factors with a dual association with DDR and centromeres. We also suggest a connection between specific DNA repair factors and the age-related increase of chromosome instability.