Corruption of DNA End-Joining in Mammalian Chromosomes by Progerin Expression

In the face of daily damage to DNA, the ability of mammalian cells to maintain a stable genome is dependent on the functionality of a variety of DNA repair pathways. Among the various forms of damage that must be dealt with is the DNA double-strand break (DSB). DSBs can be generated spontaneously in numerous ways, including processing of a variety of DNA lesions and at stalled or collapsed replication forks. DSBs can also be induced by exposure to certain chemicals or radiation. It is essential that DSBs be repaired quickly and reasonably accurately to avoid potentially deleterious genetics changes that might impact organismal viability. In our current work, we used a model cell culture system to explore how the nature of DNA repair may be altered in the premature aging syndrome known as Hutchinson-Gilford Progeria Syndrome (HGPS) [reviewed in 1].

To mend DSBs, mammalian cells utilize two types of repair pathways: homologous recombination (HR) and non-homologous end-joining (NHEJ) [reviewed in [2], [3], [4], [5], [6], [7], [8], [9], [10], [11]]. Although there are numerous sub-pathways for HR and NHEJ, the critical difference between these repair strategies is that HR utilizes a template sequence to maintain or restore genetic information at the DSB site that may otherwise be altered, whereas NHEJ involves no template in the rejoining of DNA ends. Additionally, HR is active primarily during the late S or G2 stage of the cell cycle in dividing cells, whereas NHEJ is active throughout the cell cycle and in non-dividing cells.

For a mammalian genome to remain stable, DNA repair pathways must be executed with high accuracy. However, DNA repair pathways are not foolproof. The potential exists for HR-generated genomic instability should HR occur at abnormally high levels, which may lead to increased loss-of-heterozygosity of deleterious alleles [12], [13]. Genetic perturbations that allow the choice of inappropriate recombination partners can also lead to genomic instability in the form of localized sequence alterations or chromosomal rearrangements or translocations [11], [14], [15]. Mammalian cells normally exert stringent control over HR, allowing exchange to occur only between those sequences that exhibit a very high degree of sequence identity [16], [17], [18].

Although NHEJ does not involve a template for repair and thus may produce sequence deletions or insertions, a significant portion of end-joining events normally proceeds precisely with no alteration to the sequences at the break site [19], [20], [21], [22], [23]. The balance between precise end-joining (PEJ) versus imprecise end-joining (IEJ), the size of any sequence deletions or insertions associated with IEJ, along with the appropriate regulation of HR, are all germane to the maintenance of genome stability.

The consequences of the failure of DNA transactions to maintain genome stability are varied. Aberrant HR or DSB repair is often associated with cancer [24], [25], [26]. Genomic instability is also viewed as a possible basis for, or at least a significant contributor to, the aging process. Increased levels of DNA damage, mutation, and large-scale chromosomal alterations including translocations, insertions and deletions have been observed with increasing age in humans and other organisms [27], [28], [29], [30], [31], [32], [33], [34], [35], [36], [37], [38], [39], [40]. As genome integrity is diminished over time, critical cellular functions would be expected to be disturbed. In addition, cell number would gradually be reduced as cells are lost due to apoptotic responses to unrepaired DNA lesions, leading to tissue depletion and loss of biological functions.

Much evidence has been reported that the increase in genomic instability that accompanies aging correlates with a decrease in the intrinsic efficiency of or alteration of the nature of a variety of DNA repair pathways [27], [28], [29], [30], [31], [32], [33], [34], [35], [36], [37], [38], [39], [40]. Alterations in NHEJ were reported in rat brain during aging [33], and studies with mice have suggested that the fidelity of DSB repair diminishes with age [34]. Both the efficiency and fidelity of DNA end-joining has been observed to decrease as human fibroblasts approach senescence [35]. Chromosomal DSBs accumulate in human cells approaching senescence, and it has been suggested that DSBs may be involved directly in the induction of senescence [27]. In short, evidence abounds for a role for impaired or altered repair of DSBs in the biology of aging.

Genetic disorders that produce clinical features of premature aging (progeria) are often associated with innate DNA repair defects and associated genomic instability [28], [29], [30], [31], [32], [36], [37], [38], [39], [40]. HGPS is one such rare genetic syndrome that leads to accelerated aging [1]. The average lifespan of an individual with HGPS is about fourteen years. The most common cause of HGPS is a point mutation in the LMNA gene which normally codes for lamin A and its splice variant lamin C. The mutation responsible for HGPS leads to increased usage of a cryptic splice site which leads to the production of a truncated form of lamin A referred to as "progerin." Significantly, it has been learned that progerin is in fact expressed at low levels in healthy individuals and appears to play a role in the normal aging process [41], [42], [43], [44]. Unlike wild-type fully processed lamin A, progerin retains a farnesyl group at its carboxy terminus. This farnesyl group causes progerin to largely remain associated with the inner nuclear membrane rather than localize to the nuclear lamina where lamin A normally resides.

Lamin A serves as an important component of the nuclear lamina which plays structural as well as catalytic roles in the nucleus. In HGPS, the impact of progerin over-expression on nuclear architecture is severe. The nuclei of HGPS cells are characteristically misshapen and display blebs and invaginations, and this altered nuclear structure conveys changes to numerous nuclear functions. Progerin expression in HGPS interferes with recruitment of replications factors to replication forks and this leads to replication fork stalling and collapse [40], [44], [45], [46], [47], [48]. A body of literature also implicates lamin A and its variants in DNA repair [49], [50], [51], [52], [53], [54], [55].

One important consequence of progerin expression in HGPS cells is an accumulation of DSBs and marked sensitivity to DNA damaging agents [45], [46], [47], [48], [56], [57]. The general conclusion that has been drawn is that DSB repair in HGPS is delayed or perhaps sometimes precluded, and this corruption of DNA repair is related to reduced lifespan of cells and individuals. It has also been reported that recruitment of DSB repair proteins, particularly those involved in HR repair (including Rad 50, Rad51, NBS1, and MRE11), to sites of chromosomal damage is delayed in HGPS [45], [57]. This delay in recruitment of HR-associated proteins presents an explanation for observations suggesting that NHEJ is enhanced while HR is concomitantly reduced by progerin expression [51], [52], [54], [58]. Recently, we confirmed these latter studies by directly demonstrating that progerin expression shifts repair pathway choice at a defined genomic DSB away from the HR pathway and towards NHEJ [59].

As noted above, the characteristics of end-joining including the balance between PEJ versus IEJ events are factors expected to contribute to the maintenance of genome stability. It is thus of considerable interest to explore whether progerin expression not only shifts DSB repair toward DNA end-joining, but whether progerin additionally alters the nature of DNA end-joining. In the current work, we used a novel model experimental system in mouse fibroblasts and show that progerin expression brings about a marked decrease in the frequency of PEJ with a concomitant increase in IEJ. Our data also suggest that progerin expression correlates with a possible increase in deletion size associated with DNA end-joining. Additional experiments show that progerin expression does not allow genetic exchange between imperfectly matched sequences and thus, while reducing the occurrence or HR, progerin does not decrease the fidelity of HR. Collectively, our work suggests that progerin expression compromises genome stability by corrupting interactions between complementary sequences at DNA termini during DNA repair. Such influences of progerin may impact genome stability and contribute to the aging process.

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