Fanconi anaemia syndrome and apoptosis: state of the art

Fanconi anemia (FA) is a rare recessive, human genetic syndrome characterized by progressive bone marrow failure, developmental abnormalities, predisposition to malignancy, chromosomal instability and DNA damage hypersensitivity. Two (FAA and FAC) of the five genes involved were cloned but their functions remain unknown. At present, the involvement of FA proteins in DNA repair, redox status of the cell and apoptosis are areas of intensive investigation. The aim of this review is to synthesize current results and ideas concerning the involvement of apoptosis in the FA phenotype and conversely, the role of FA proteins in the control of apoptosis.     

A FANCD2 domain activates Tip60‐dependent apoptosis

The FA (Fanconi anaemia) FANCD2 protein is pivotal in the cellular response to DNA interstrand cross‐links. Establishing cells expressing exogenous FANCD2 has proven to be difficult compared with other DNA repair genes. We find that in transformed normal human fibroblasts, exogenous nuclear expression of FANCD2 induces apoptosis, dependent specifically on exons 10–13. This is the same region required for interaction with the histone acetyltransferase, Tip60. Deletion of exons 10–13 from FANCD2 N‐terminal constructs (nucleotides 1–1100) eliminates the binary interaction with Tip60 and the cellular apoptotic response; moreover, cells can stably express FANCD2 at high levels if Tip60 is depleted. The results indicate that FANCD2‐sponsored apoptosis requires an interaction with Tip60 and depends on Tip60.     

The Effects of Free Radicals on Cobalamin and Iron

The aim of this communication is to show the means by which free radicals could deleteriously alter the metabolism of cobalamin (vitamin B12) and iron in their attempt to protect the body against neoplasia or inflammation and in doing so, create the anaemia of chronic disease.     

Environment change, geographic migration and sickle cell anaemia

The classic model describing the interaction between sickle cell anaemia and malaria is one of the most notable achievements of population genetics. Nevertheless, only panmictic populations in steady environments have been studied theoretically to date. In this paper, environment change and geographic inhomogeneity are introduced. The rate of decrease of mutation after environment improvement is obtained. The kinetics of the spread of disease after the initial mutation, together with the genetic composition profile near the borders of malaria areas, are calculated. The results are compared with the empirical data on the mutation level in African and African-American populations. It is shown that the spread of disease and decrease in mutation are highly asymmetric: the mutation level increases exponentially and decreases much more slowly (as a power function). The mathematical and biological reasons for this behaviour are discussed.     

MHF1 plays Fanconi anaemia complementation group M protein (FANCM)‐dependent and FANCM‐independent roles in DNA repair and homologous recombination in plants

Fanconi anaemia complementation group M protein (FANCM), a component of the human Fanconi anemia pathway, acts as DNA translocase that is essential during the repair of DNA interstrand cross‐links. The DNA‐damage‐binding function of FANCM is strongly enhanced by the histone fold‐containing FANCM‐associated protein MHF1. We identified a single homologue of MHF1 in the genome of Arabidopsis thaliana. Similar to the loss of AtFANCM, the loss of AtMHF1 leads to several meiotic defects, such as chromosome bridges between bivalents and an unequal distribution of chromosomes. Moreover, MHF1, together with FANCM, is involved in interstrand cross‐link repair in plants. This phenotype is detectable only in double mutants of the RecQ helicase and BLM homologue RECQ4A, which appears to function in a parallel pathway to the FANCM/MHF1 complex. However, in somatic cells, FANCM has an MHF1‐independent function in replicative repair in a parallel pathway to the endonuclease MUS81. Furthermore, MHF1 is required for efficient somatic homologous recombination (HR) – a role antagonistic to FANCM. FANCM and RECQ4A define two parallel pathways of HR suppression in Arabidopsis. Hyperrecombination in the fancm but not the recq4A mutant can be abolished by MHF1 mutations. This finding indicates that MHF1 and FANCM act at different steps of a single, common, HR pathway.     

Structural and biochemical basis of interdependent FANCI‐FANCD2 ubiquitination

Di‐monoubiquitination of the FANCI‐FANCD2 (ID2) complex is a central and crucial step for the repair of DNA interstrand crosslinks via the Fanconi anaemia pathway. While FANCD2 ubiquitination precedes FANCI ubiquitination, FANCD2 is also deubiquitinated at a faster rate than FANCI, which can result in a FANCI‐ubiquitinated ID2 complex (I_UbD2). Here, we present a 4.1 Å cryo‐EM structure of I_UbD2 complex bound to double‐stranded DNA. We show that this complex, like ID2_Ub and I_UbD2_Ub, is also in the closed ID2 conformation and clamps on DNA. The target lysine of FANCD2 (K561) becomes fully exposed in the I_UbD2‐DNA structure and is thus primed for ubiquitination. Similarly, FANCI''s target lysine (K523) is also primed for ubiquitination in the ID2_Ub‐DNA complex. The I_UbD2‐DNA complex exhibits deubiquitination resistance, conferred by the presence of DNA and FANCD2. ID2_Ub‐DNA, on the other hand, can be efficiently deubiquitinated by USP1‐UAF1, unless further ubiquitination on FANCI occurs. Therefore, FANCI ubiquitination effectively maintains FANCD2 ubiquitination in two ways: it prevents excessive FANCD2 deubiquitination within an I_UbD2_Ub‐DNA complex, and it enables re‐ubiquitination of FANCD2 within a transient, closed‐on‐DNA, I_UbD2 complex.     

Comparative action of calpain on erythrocyte Ca2+-pumping ATPase in sickle cell anaemia,essential hypertension and kwashiorkor

Calpain, a calcium-dependent, neutral cysteine-protease was purified from the erythrocyte cytosol of subjects having essential hypertension (HTN), sickle cell anaemia, (SCA), or kwashiorkor (KWA). Identical electrophoretic mobility on SDS-polyacrylamide gradient gel, sensitivity to micromolar amounts of Ca²⁺, absolute requirement for a reducing environment and a high susceptibility to inhibition by leupeptin and thiol-group modifying reagents confirm that calpain preparations from these erythrocytes are equivalent to calpain I. Whereas the extent of calpain activation of erythrocyte membrane Ca²⁺-pumping ATPase of normal subjects was almost equal to that due to calmodulin, calpain activation of the HTN and SCA pump was greater than activation by calmodulin. Like in normal membranes, exogenous calmodulin protected the Ca²⁺-pumping ATPase of these erythrocytes against calpainization; the degree of protection by calmodulin is least in SCA and HTN. Electrophoretic separation of erythrocyte membranes and the purified Ca²⁺-pumping ATPase of HTN, SCA and KWA subjects does not indicate the presence of fragments resulting from the proteolytic action of calpain.Abbreviations PMSF phenylmethylsulfonylfluoride - TLCK N--tosyl-L-lysine chloromethyl ketone - EGTA ethyleneglycol-bis (-aminoethylether) N,N¹-tetraacetic acid - ATP adenosine 5¹-triphosphate - Hepes 4-(2 hydroxyethyl)-1-piperazine ethanesulphonic acid - Tris-HC1 Tris (hydroxymethyl) aminomethane-hydrochloride - SDS sodium dodecyl sulphate     

Inherited haemolytic anaemia created by insertional inactivation of the α-spectrin gene

In the process of generating transgenic mice, inserted foreign DNA can cause insertional inactivation of the flanking genetic locus and simultaneously provide a molecular tag for localizing and cloning the inactivated gene. We describe the case of an insertional mutation leading, in animals homozygous for the insertion, to severe anaemia that was lethal within a few days after birth. The haemolytic anaemia and microspherocytosis of the red cells strongly suggested membrane abnormalities of the erythrocytes. Byin situ localization of the integration site, protein analysis of the red cell membranes, northern and Southern blot analyses, we were able to demonstrate that the integrated transgene had affected the α-spectrin gene locus.