Causes and Frequencies of Double-Strand Breaks
There are an estimated ten double-strand breaks (DSBs) per day per cell, based on metaphase chromosome and chromatid breaks in early passage primary human or mouse fibroblasts (11-13). Estimates of DSB frequency in nondividing cells are difficult to make because methods for assessing DSBs outside of metaphase are subject to even more caveats of interpretation.
In mitotic cells of multicellular eukaryotes, DSBs are all pathologic (accidental) except the specialized subset of physiologic DSBs in early lymphocytes of the vertebrate immune system (Fig. 1). Major pathologic causes of double-strand breaks in wild type cells include replication across a nick, giving rise to chromatid breaks during S phase. Such DSBs are ideally repaired by HR using the nearby sister chromatid.
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All of the remaining pathologic forms of DSB are repaired by NHEJ because they usually occur when there is no nearby homology donor and/or because they occur outside of S phase. These causes include reactive oxygen species from oxidative metabolism, ionizing radiation, and inadvertent action of nuclear enzymes (14).
Reactive oxygen species (ROS) are a second major cause of DSBs (Fig. 1). During the course of normal oxidative respiration, mitochondria convert about ~0.1 to 1% of the oxygen to superoxide (O-2) (15). Superoxide dismutase in the mitochondrion (SOD2) or cytosol (SOD1) can convert this to hydroxyl free radicals, which may react with DNA to cause single-strand breaks. Two closely spaced lesions of this type on anti-parallel strands can cause a DSB. About 1022 free radicals or ROS species are produced in the human body each hour, and this represents about 109 ROS per cell per hour. A subset of the longer-lived ROS may enter the nucleus via the nuclear pores.
A third cause of DSBs is natural ionizing radiation of the environment. These include gamma rays and X-rays. At sea level, ~300 million ionizing radiation particles per hour pass through each person. As these traverse the body, they create free radicals along their path, primarily from water. When the particle comes close to a DNA duplex, clusters of free radicals damage DNA, generating double- and single-stranded breaks at a ratio of about 25 to 1 (16). About half of the ionizing radiation that strikes each of us comes from outside the earth. The other half of the radiation that strikes us comes from the decay of radioactive elements, primarily metals, within the earth.
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A fourth cause of DSBs is inadvertent action by nuclear enzymes on DNA. These include failures of type II topoisomerases, which transiently break both strands of the duplex. If the topoisomerase fails to rejoin the strands, then a DSB results (17). Inadvertent action by nuclear enzymes of lymphoid cells, such as the RAG complex (composed of RAG1 and 2) and activation-induced deaminase (AID) are responsible for physiologic breaks for antigen receptor gene rearrangement; however, they sometimes accidentally cleave the DNA at off-target sites outside the antigen receptor gene loci (18). In humans, these account for about half of all of the chromosomal translocations that result in lymphoma.
Finally, physical or mechanical stress on the DNA duplex is a relevant cause of DSBs. In prokaryotes, this arises in the context of desiccation, which is quite important in nature (19). In eukaryotes, telomere failures can result in chromosomal fusions that have two centromeres, and this results in physical stress by the mitotic spindle (breakage/fusion/bridge cycles) with DSBs (20).
In addition to the above for mitotic cells, meiotic cells have an additional source of DSBs, which is physiologic and is caused by an enzyme called Spo11, a topoisomerase II-like enzyme (21). Spo11 creates DSBs to generate cross-overs between homologues during meiotic prophase I. These events are resolved by HR. Therefore, NHEJ is not relevant to Spo11 breaks. Interestingly, it is not clear that NHEJ occurs in vertebrate meiotic cells, because one group reports the lack of Ku70 in spermatogonia (22). Human spermatogonia remain in meiotic prophase I for about 3 weeks, and human eggs remain in meiotic prophase I for 12 to 50 years; hence, these cells can rely on HR during these long periods. Given the error-prone nature of NHEJ (see below), reliance on HR may be one way to minimize alterations to the germ line at frequencies that might be deleterious to a population.
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