Clues to how cells repair broken DNA
Our genetic material is stored in the nucleus of each of our cells — protected from constant environmental and metabolic assault. But over a life-time, DNA will suffer damage. Although cells have a host of ways to deal with injury, sometimes things go wrong.
Agata Smogorzewska MD PhD, Associate Professor and head of the
Laboratory of Genome Maintenance at Rockefeller University, New York, USA, studies how cells repair interstrand crosslinks (ICL), a particular type of damage to DNA in which both double helix strands are physically stuck together. This prevents DNA from replicating and genes from being expressed into proteins — the two basic functions of DNA [see article 4/20/16}.
Repair of these lethal crosslinks takes place in DNA replication and requires cutting out both cross-linked bases. This is accomplished in a multi-step process in the rare disorder Fanconi anemia (FA) where cell components promote Homologous Recombination (HR), a process cells use to repair double-strand breaks. FA patients suffer from bone marrow failure and infertility presumed to be due to their inability to form and maintain specific types of blood cells.
Fanconi anemia (FA) arrises prenatally. Blood cell formation occurs in the yolk sack, later in the liver, and finally in the bone marrow. FA is also associated with a very high incidence of cancers, most likely due to incorrect repair of ICL breaks. Inducing ICL repair is a major cancer treatment.
In her most recent paper published in Genes and Development, Smogorzewska and her team engineered mice lacking one of the genes involved in ICL repair. These mice turn out to be particularly susceptible to drugs that induce DNA cross-links, as their cells don't have a functional DNA repair system. Using these mice, researchers will now be able to define more mechanisms involved in DNA repair.
Eliminating genes in mice is a standard laboratory technique for determining what those genes' function would have been. They are called knockout mice, for the genes they have lost. This study was called FAN1 after the Fanconi anemia pathway.
Mutations in many of the genes regulating DNA interstrand cross-link (ICL) repair cause Fanconi anemia. To understand why, Smogorzewska searched to identify those genes. Unexpectedly, she found one that causes a completely different disease.
In humans, FAN1 deficiency causes karyomegalic interstitial nephritis, a disease leading to kidney failure at around age 30. So far, the connection between the gene, which produces a DNA-cutting enzyme called a nuclease, and the kidney disease resulting from its loss is still obscure. FAN1 knockout mice will now allow reearchers to investigate FAN1's role in DNA repair and how its mutation leads to kidney failure.
One observation in kidney cells from FAN1 knockout mice, is their cell nuclei are abnormally large — karyomegaly — as seen in patients with karyomegalic interstitial nephritis (KTN). KTN is thought to occur following too many DNA duplications.
Scientists also saw enlarged nuclei in livers of FAN1 knockout mice with these mice later developing liver disease. Perhaps FAN1 protein helps protect liver and kidney function as animals age?
Smogorzewska: "Both the kidney and the liver need to deal with a lot of toxins. FAN1 helps them do their job of detoxification. FAN1 interacts with many different components in the cell nucleus and might be implicated in more genetic processes than what we previously thought."
Although FAN1 is clearly necessary for interstrand cross-link (ICL) repair, researchers believe it is possible that kidney and liver dysfunction are not directly due to repair malfunctions. Although FAN1 interacts with DNA repair proteins, implicated in Fanconi anemia, this study demonstrates it actually doesn't have to do so in order to repair DNA damage. This might explain why karyomegalic interstitial nephritis and Fanconi anemia cause completely different symptoms in patients.
Smogorzewska: "We don't know what FAN1 actually does in the cell — what activates it, what it interacts with, and the specific type of DNA damage it repairs. We don't know what it is seeing that other nucleases aren't seeing."
Smogorzewska's lab now wants to explore how the FAN1 gene knockout affects kidney degeneration. Initially, her lab will identify the toxins causing DNA cross-links and then why patients with karyomegalic interstitial nephritis can't self repair.
Environmental toxins, including some herbal medicines known to cause kidney disease, don't seem to be involved. So internal toxins — normal byproducts of metabolism — are the most likely culprits.
Smogorzewska plans to test how FAN1 repairs DNA damaged by formaldehyde and acetaldehyde, toxins produced in the body which induce DNA cross-links.
Deficiency of FANCD2/FANCI-associated nuclease 1 (FAN1) in humans leads to karyomegalic interstitial nephritis (KIN), a rare hereditary kidney disease characterized by chronic renal fibrosis, tubular degeneration, and characteristic polyploid nuclei in multiple tissues. The mechanism of how FAN1 protects cells is largely unknown but is thought to involve FAN1's function in DNA interstrand cross-link (ICL) repair. Here, we describe a Fan1-deficient mouse and show that FAN1 is required for cellular and organismal resistance to ICLs. We show that the ubiquitin-binding zinc finger (UBZ) domain of FAN1, which is needed for interaction with FANCD2, is not required for the initial rapid recruitment of FAN1 to ICLs or for its role in DNA ICL resistance. Epistasis analyses reveal that FAN1 has cross-link repair activities that are independent of the Fanconi anemia proteins and that this activity is redundant with the 5′–3′ exonuclease SNM1A. Karyomegaly becomes prominent in kidneys and livers of Fan1-deficient mice with age, and mice develop liver dysfunction. Treatment of Fan1-deficient mice with ICL-inducing agents results in pronounced thymic and bone marrow hypocellularity and the disappearance of c-kit+ cells. Our results provide insight into the mechanism of FAN1 in ICL repair and demonstrate that the Fan1 mouse model effectively recapitulates the pathological features of human FAN1 deficiency.
Received December 9, 2015.
Accepted February 9, 2016.
© 2016 Thongthip et al.; Published by Cold Spring Harbor Laboratory Press
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