Abstract. Adducin is a membrane-skeletal protein which is a candidate to promote assembly of a spectrin-actin network in erythrocytes and at sites of cellcell contact in epithelial tissues . The complete sequence of both subunits of human adducin, alpha (737 amino acids), and beta (726 amino acids) has been deduced by analysis of the cDNAs . The two subunits have strikingly conserved amino acid sequences with 49% identity and 66% similarity, suggesting evolution by gene duplication . Each adducin subunit has three distinct domains: a 39-kD NH2-terminal globular protease-resistant domain, connected by a 9-kD domain to a 33-kD COOH-terminal protease-sensitive tail comprised almost entirely of hydrophilic amino acids . The tail is responsible for the high frictional ratio of adducin noted previously, and was visualized by EM . The head domains of both adducin subunits exhibit a limited sequence similarity with the NH2-terminal ac-LASMA membranes of eukaryotic cells contain a system of structural proteins known as the spectrin-based membrane skeleton that has a general role in organi zation ofcertain integral membrane proteins and of coupling these proteins to cytoplasmic proteins (2,16,29) . Spectrin, the principal component of the membrane skeleton, is a rodshaped protein that associates with F-actin ateach end . Spectrin and actin together with accessory proteins form a regular geodesic domelike structure in erythrocytes where short actin filaments are associated with five and seven spectrin molecules to form a polygonal network (5, 28) . The striking images of the spectrin skeleton raise the issue of how such a structure could be assembled and has focused attention on the accessory proteins that interact with spectrin and actin at spectrin-actin junctions (1) .Adducin is a membrane-skeletal protein that promotes association of spectrin with actin, and this interaction is regulated by calcium/calmodulin (14, 31) . Adducin is comprised of two subunits with M 103,000 (alpha) and 97,000 (beta) and was originally purified from human erythrocytes based on its calmodulin-binding activity (13) . Adducin also is a substrate for protein kinase C in intact cells as well as under in vitro conditions (6,27,33,39). A protein closely related

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DNA damage is a hallmark of cancer, and mutation and misregulation of proteins that maintain genomic fidelity are associated with the development of multiple cancers. DNA double strand breaks are arguably considered the most deleterious type of DNA damage. The nonhomologous end-joining (NHEJ) pathway is one mechanism to repair DNA double strand breaks, and proteins involved in NHEJ may also regulate DNA replication. We previously established that DNA-PKcs, a NHEJ protein, promotes genomic stability and cell viability following cellular exposure to replication stress; we wanted to discern whether another NHEJ protein, DNA ligase IV (Lig4), shares this phenotype. Our investigations focused on triple negative breast cancer cells, as, compared to nonbasal breast cancer, LIG4 is frequently amplified, and an increased gene dose is associated with higher Lig4 expression. We depleted Lig4 using siRNA and confirmed our knockdown by qPCR and western blotting. Cell survival diminished with Lig4 depletion alone, and this was associated with increased replication fork stalling. Checkpoint protein Chk1 activation and dephosphorylation were unchanged in Lig4-depleted cells. Lig4 depletion resulted in sustained DNA-PKcs phosphorylation following hydroxyurea exposure. Understanding the effect of Lig4 on genomic replication and the replication stress response will clarify the biological ramifications of inhibiting Lig4 activity. In addition, Lig4 is an attractive clinical target for directing CRISPR/Cas9-mediated repair towards homology-directed repair and away from NHEJ, thus understanding of how diminishing Lig4 impacts cell biology is critical.