DNA repair/pro-apoptotic dual-role proteins in five major DNA repair pathways: fail-safe protection against carcinogenesis

Abstract

Two systems are essential in humans for genome integrity, DNA repair and apoptosis. Cells that are defective in DNA repair tend to accumulate excess DNA damage. Cells defective in apoptosis tend to survive with excess DNA damage and thus allow DNA replication past DNA damages, causing mutations leading to carcinogenesis. It has recently become apparent that key proteins which contribute to cellular survival by acting in DNA repair become executioners in the face of excess DNA damage.

Five major DNA repair pathways are homologous recombinational repair (HRR), non-homologous end joining (NHEJ), nucleotide excision repair (NER), base excision repair (BER) and mismatch repair (MMR). In each of these DNA repair pathways, key proteins occur with dual functions in DNA damage sensing/repair and apoptosis. Proteins with these dual roles occur in: (1) HRR (BRCA1, ATM, ATR, WRN, BLM, Tip60 and p53); (2) NHEJ (the catalytic subunit of DNA-PK); (3) NER (XPB, XPD, p53 and p33^ING1b); (4) BER (Ref-1/Ape, poly(ADP-ribose) polymerase-1 (PARP-1) and p53); (5) MMR (MSH2, MSH6, MLH1 and PMS2). For a number of these dual-role proteins, germ line mutations causing them to be defective also predispose individuals to cancer. Such proteins include BRCA1, ATM, WRN, BLM, p53, XPB, XPD, MSH2, MSH6, MLH1 and PMS2.

Introduction

A widely held view is that the initial reaction of a cell to DNA damage is to repair the damage. However, with increasing levels of DNA damage the cell switches to cell cycle arrest or to apoptosis. Cell cycle arrest is sometimes permanent, but ordinarily reversible, allowing time for further DNA repair. The maintenance of a switching mechanism that shifts the cell from DNA repair to apoptosis, as appropriate in the presence of excessive DNA damage, appears to be of central importance for avoiding progression to cancer. The default mechanism of apoptosis prevents clonal expansion of cells in which unrepaired damage would lead to mutation and to carcinogenesis (Fig. 1).

During progression to cancer, the capability of cells to undergo apoptosis is often reduced, suggesting that the normal ability to undergo apoptosis is protective against cancer. For example, loss of the capacity to undergo apoptosis occurs early in the progression to adenocarcinoma of the colon [1], [2], [3] and esophagus [4], [5]. Apoptosis capability may be reduced in three ways: by mutation, by silencing or loss of genes encoding required components of the apoptosis pathway (e.g. p53 and bax) [6], [7], or by persistent activation of genes encoding apoptosis suppressors (e.g. bcl-2) [8]. Failure to undergo apoptosis in the face of unrepaired damage leads to enhanced mutation [9], [10], [11], including chromosome aberrations, and can be a cause of the genomic instability that is a general characteristic of cancer progression [12].

In the following sections, we discuss the various specific DNA repair processes, how these processes recognize and repair DNA damage, and how they switch from repair to apoptosis when DNA damage presumably overwhelms repair capacity. One should keep in mind, however, that of necessity most of the mechanistic information provided is based on in vitro or purified enzyme studies and, thus, may or may not reflect processes in vivo in man. Similarly, biochemical events observed in cultured cells or with purified enzymes may provide valuable clues to in vivo processes, but may not be exactly correct.

There are two distinct types of cell death in vivo, apoptosis and necrosis [13], [14], [15]. Apoptosis is a controlled form of cell death, in which the cell undergoes “cellular suicide”. The cell shrinks, dehydrates, fragments its nucleus, and is phagocytized by macrophages [16]. Necrosis, on the other hand, is a traumatic, but passive, form of cell death, in which ion pumps fail, the cell swells and then undergoes lysis with the release of inflammatory mediators [13], [17].

A cell will first try to repair any DNA damage and survive; however, if DNA damages are excessive, the preferred mode of cell death in a multicellular organism is apoptosis, a process which does not elicit an inflammatory response. How does a cell ensure that its death will occur by apoptosis, rather than necrosis? Given that both DNA repair and apoptosis are energy-demanding processes, the answer may lie in the proper utilization of the available ATP in the cell (Fig. 2) [18], [19], [20]. Energy is required for both DNA repair [21], [22], [23], [24], [25], [26], [27], [28], [29], [30], [31], [32], [33], [34], [35] and apoptosis [18], [36], [37], [38], [39], [40]. In addition, during both DNA repair and apoptosis, the ATP-dependent ion pumps that keep Ca⁺⁺ [41] at a low cytosolic concentration and sustain a critical internal K⁺ concentration [42] need to be maintained.

If the repair of DNA damage is prolonged in any given cell, an “energy catastrophe” [43] will occur (Fig. 2). The considerable investment of energy to repair DNA was particularly noted by Roca and Cox [25] in bacteria, where they pointed out the “profligate” chemical energy invested in the degradation and replacement of a strand of DNA, 1000 bases or more in length, to repair one DNA mismatch. Therefore, in mammalian cells, a large amount of ATP may be similarly directed toward the repair of DNA and diverted away from the ATP-dependent steps required for the execution phase of apoptosis. In particular, the formation of the apoptosome [44], [45], [46], [47], [48], [49], the multimeric complex consisting of dATP, Apaf-1 and cytochrome c necessary for the formation of active caspase-9 [50], and the activation of downstream effector caspases (e.g. caspase-3, -6 and -7) during the demolition phase of apoptosis [46], [49], [51], require dATP or ATP [36], [37]. In addition, in the face of excessive DNA damage, ATP is diverted from maintaining the ion pumps (Fig. 2). In the case of the Ca⁺⁺-ATPase, energy depletion prevents Ca⁺⁺ ions from being extruded from the cell, thereby increasing the intracellular Ca⁺⁺ concentration from nanomolar to lethal micromolar concentrations. The influx of excessive Ca⁺⁺ activates calcium-dependent phospholipases, nucleases and proteases, which dramatically injure the cell. Failure of the Na⁺, K⁺-ATPase causes excessive amounts of Na⁺ to enter the cell, followed by a large influx of water, resulting in cellular swelling and lysis. Since K⁺ normally prevents apoptosis [52] by inhibiting the Apaf-1 oligomerization step in the formation of the apoptosome [53], the efflux of K⁺ from the cell will help shift the mode of cell death from apoptosis to necrosis. It may be that, in order to ensure that apoptosis occurs instead of necrosis in the face of excessive DNA damage, key proteins involved in DNA repair that are consumers of energy are cleaved and inactivated during apoptosis, such as ATM [54], [55], DNA-PK [56], hsRad51 [57], [58], [59] and PARP [60], [61], [62], [63]. Inactivation of these proteins diverts ATP from DNA repair, and may also prevent confusing pro-survival signals. In addition to the formation of the apoptosome, ATP is also necessary for the accumulation and stabilization of p53 [27], a DNA damage-responsive transcription factor that increases the expression of pro-apoptotic proteins [64] (e.g. bax) which damage the mitochondrial outer membrane with the release of cytochrome c (Fig. 2) [65]. p53 also increases the expression of Apaf-1 [66], [67], which is part of the apoptosome. During DNA damage-induced apoptosis, p53 has also been reported to directly target the mitochondria [47].

The cleavage of PARP during apoptosis appears to be an especially critical step, since PARP rapidly consumes NAD⁺, the pyridine nucleotide, which, in its reduced form, contributes electrons to complex I of the mitochondrial electron transport chain (Fig. 2). The NAD⁺ precursors, nicotinic acid and nicotinamide, in fact, can protect against apoptosis induced by the multiple stress-inducer, deoxycholate [68]. Since PARP also poly(ADP) ribosylates and inhibits p53 [69], PARP cleavage will result in the activation of p53 (Fig. 2). The cleavage of PARP also results in a gain-of-function, which augments the loss of PARP activity [70]. After cleavage by caspase-3, the N-terminal apoptotic fragment of PARP retains a strong DNA binding activity and totally inhibits the catalytic activity of uncleaved PARP [70]. Fig. 2 outlines a mechanism whereby the cell senses the presence of too much DNA damage, and shunts the available ATP toward apoptosis, while at the same time, maintaining ion pumps that prevent cellular lysis. In vivo, cell fate will culminate with the phagocytosis of the apoptotic cell and/or apoptotic bodies. In vitro, cell fate will culminate in the eventual loss of ion pumps, a fate termed “secondary necrosis” of apoptotic cells. Since phagocytes are usually absent from most in vitro culture systems, phagocytosis of early apoptotic cells does not occur.

Over time, a high level of apoptosis can lead to clonal selection of apoptosis-resistant cells. The generation of mutations as a consequence of increased unrepaired DNA damage in apoptosis-resistant, proliferating cells appears to be an important aspect of the development of cancer at numerous sites within the body. This is exemplified by the natural progression of follicular lymphoma to high-grade lymphoma [71]. The constitutive presence of bcl-2 confers apoptosis resistance on follicular lymphoma cells, which then allows mutations and chromosomal aberrations to increase. As reviewed next, the mechanism of apoptosis resistance involves, in part, the loss of key bi-functional proteins which are necessary for initiation of both apoptosis and DNA repair. Loss of apoptosis competence coupled with loss of capability to repair DNA damage increases genomic instability which in turn accelerates progression to cancer (Fig. 1) [72].

Five major DNA repair pathways are homologous recombinational repair (HRR); non-homologous end joining (NHEJ); nucleotide excision repair (NER); base excision repair (BER); and mismatch repair (MMR) [73]. We review evidence that key proteins associated with these five major forms of DNA repair also have a role in triggering cell cycle arrest and apoptosis (Table 1).