DNA topoisomerase 2-binding protein 1 (TOPBP1) is a scaffold protein that in humans is encoded by the TOPBP1 gene.

TOPBP1 was first identified as a protein binding partner of DNA topoisomerase-IIβ by a yeast 2-hybrid screen, giving it its name. TOPBP1 is involved in a variety of nuclear specific events. These include DNA damage repair, DNA replication, transcriptional regulation, and cell cycle checkpoint activation. TOPBP1 primarily regulates the DNA damage repair response through its ability to activate the damage response kinase, ataxia-telangiectasia mutated and RAD3-related (ATR). It also plays a critical role in DNA replication initiation and regulation of the cell cycle. Changes in TOPBP1 gene expression are associated with pulmonary hypertension, breast cancer, glioblastoma, non-small cell lung cancer, and sarcomas.

The TOPBP1 gene encodes a scaffold protein which facilitates interactions between different proteins at specific times and locations. It accomplishes these interactions with other protein partners through its breast cancer associated gene 1 C-terminus (BRCT) domains. A BRCT domain is structurally defined by a 4 member β sheet that is bookended by one α-helix (α2) and two other α-helices (α1 and α3). The amino acid residues that make up these core features are highly conserved, with protein specific deviations occurring in the loops that connect these subunits. BRCT domains canonically act in pairs, with one domain acting as the acceptor for phosphorylated binding partners and the other domain possessing a binding motif that provides specificity. These pairs are separated by a linker sequence that varies by protein. The paired domains associate through hydrophobic packing interactions that occur between the N-terminal BRCT domain's α2 helix and the C-terminal BRCT domain's α1 and α3 helices. These interactions facilitate BRCT domain binding with phosphorylated binding partners. In contrast, BRCT domains can also exist as either single domains or as a fusion of two different domains.

Human TOPBP1 has nine unique BRCT domains, with four conserved from the budding yeast homologue Dpb11 (i.e. BRCT1,2 and BRCT4,5). In human TOPBP1 the BRCT0, BRCT1, and BRCT2 domains uniquely exist in triple domain form, which is in contrast to the yeast Dpb11 canonical double domain. Only the BRCT3 and BRCT6 domains exist as single domains and may not be able to bind phosphoprotein partners TOPBP1 also contains an ATR activation domain (AAD) that is located between the BRCT6 and BRCT7 domains. Through these BRCT specific interactions TOPBP1 mediates DNA damage repair, DNA replication, transcription, and mitosis.

To regulate its activity, TOPBP1 has been found to self-oligomerize at the BRCT7/8 domains, as it responds to replicative stress.

TOPBP1 was first identified as a DNA damage protein through its association with BRCA1, which is a protein heavily implicated in breast cancer pathology. TOPBP1 was found in complex with BRCA1 at sites independent from replication forks (i.e identified by the DNA replication clamp proliferating cell nuclear antigen) during normal S phase. When DNA damage was induced at higher levels by γ irradiation, there was an increase in TOPBP1/BRCA1 at sites away from replication forks. In contrast, when replication forks were stalled by hydroxyurea to generate DNA replication stress, TOPBP1/BRCA1 were found at sites of replication forks. This showed a DNA damage specific role for TOPBP1 recruitment at both replication sites and non-replication sites. To mediate these aspects of DNA repair, TOPBP1 was found to associate with Rad9, which forms a complex with Rad1 and Hus1, hereby termed the 9-1-1 DNA repair clamp. TOPBP1 binds to Rad9 with its BRCT0/1/2 domains. The BRCT1 domain was found to be directly responsible for mediating the phosphorylation dependent interaction with Rad9.

DNA damage repair is initiated and maintained by two kinases, ataxia-telangiectasia mutated (ATM) and ATR, with ATR proving to be more important for maintaining the genome. TOPBP1 has been shown to be an activator of ATR, leading to an increase in the kinase activity of ATR. Following instances of DNA damage that lead to double stranded breaks (DSBs) and subsequent repair mediated resection, there will be long sequences of single stranded DNA (ssDNA) exposed. This ssDNA will become coated with replication protein A (RPA). ATR is successfully honed to RPA coated ssDNA by ATR interacting protein (ATRIP). The junction of RPA coated ssDNA and intact double stranded DNA (dsDNA) is where TOPBP1 and the 9-1-1 clamp is recruited. In addition to TOPBP1, ATR has also been found to be activated by the ssDNA specific, RPA interacting protein ETAA1.

TOPBP1/9-1-1 recruitment is conducted independent of ATRIP/ATR which serves as a regulatory mechanism that prevents both premature and non-specific activation of the DNA damage response pathway. TOPBP1 interacts with ATR through its ATR activating domain (AAD), which is located between the BRCT domains 6 and 7. The AAD domain of TOPBP1 alone is sufficient for activating ATR kinase activity in vitro. Knockdowns of TOPBP1 gene expression leads to a reduction in phosphorylation of downstream ATR kinase targets. The specific activation mechanism of ATR is still unknown, but it is thought that TOPBP1 binding to ATR induces a conformational change that promotes catalysis above baseline kinase activity. Following ATR activation, it is able to phosphorylate downstream DNA damage associated factors, with the primary effector being the kinase Chk1.