Association of the Rad9–Rad1–Hus1 checkpoint clamp with MYH DNA glycosylase and DNA

Highlights

• Individual Rad9–Rad1–Hus1 (9–1–1) subunits play distinct roles in BER.

• The interdomain connecting loop of Hus1 is a key determinant of MYH binding.

• The K136A mutant demonstrated that Hus1 binding is uncoupled from MYH activation.

• The order of DNA binding affinity is: 9¹⁻²⁶⁶–1–1 ≥ Rad9¹⁻²⁶⁶

• Hus1 ≫ Rad1.

• Preferential binding of 9–1–1 to 5′-recessed DNA is independent of clamp loader.

Abstract

Cell cycle checkpoints provide surveillance mechanisms to activate the DNA damage response, thus preserving genomic integrity. The heterotrimeric Rad9–Rad1–Hus1 (9–1–1) clamp is a DNA damage response sensor and can be loaded onto DNA. 9–1–1 is involved in base excision repair (BER) by interacting with nearly every enzyme in BER. Here, we show that individual 9–1–1 components play distinct roles in BER directed by MYH DNA glycosylase. Analyses of Hus1 deletion mutants revealed that the interdomain connecting loop (residues 134–155) is a key determinant of MYH binding. Both the N-(residues 1–146) and C-terminal (residues 147–280) halves of Hus1, which share structural similarity, can interact with and stimulate MYH. The Hus1^K136A mutant retains physical interaction with MYH but cannot stimulate MYH glycosylase activity. The N-terminal domain, but not the C-terminal half of Hus1 can also bind DNA with moderate affinity. Intact Rad9 expressed in bacteria binds to and stimulates MYH weakly. However, Rad9¹⁻²⁶⁶ (C-terminal truncated Rad9) can stimulate MYH activity and bind DNA with high affinity, close to that displayed by heterotrimeric 9¹⁻²⁶⁶–1–1 complexes. Conversely, Rad1 has minimal roles in stimulating MYH activity or binding to DNA. Finally, we show that preferential recruitment of 9¹⁻²⁶⁶–1–1 to 5′-recessed DNA substrates is an intrinsic property of this complex and is dependent on complex formation. Together, our findings provide a mechanistic rationale for unique contributions by individual 9–1–1 subunits to MYH-directed BER based on subunit asymmetry in protein–protein interactions and DNA binding events.

Introduction

Cell cycle checkpoints provide surveillance mechanisms to activate the DNA damage response (DDR), thus preserving genomic integrity [1], [2]. Activation of DDR leads to cell cycle arrest (which allows time for DNA repair) and enhances DNA repair. When DNA damage is extreme, apoptosis is triggered. The checkpoint system includes an array of proteins that function as sensors, transducers, and effectors [3], [4], [5], [6]. The heterotrimeric Rad9/Rad1/Hus1 (9–1–1) complex is a DDR sensor [7], [8] and is loaded onto DNA by the Rad17-RFC_2–5 clamp loader [9], [10], [11], [12]. 9–1–1 is essential for embryonic development, genomic stability, and telomere integrity [13], [14], [15], [16]. Besides serving as a damage sensor [17], 9–1–1 is involved in many DNA metabolisms [14] including base excision repair (BER) (reviewed in [18]). Remarkably, 9–1–1 interacts with nearly every enzyme in BER and is proposed to constitute a platform to coordinate BER. Many BER proteins interact selectively with specific subunit(s) of 9–1–1 [19], [20], [21].

The first step in BER is carried out by a DNA glycosylase, which cleaves damaged or mismatched bases. MYH (also called MUTYH) DNA glycosylase excises adenine when it is mispaired with 8-oxo-7,8-dihydroguanine (G⁰) or guanine and thus reduces G:C to T:A mutations [22], [23], [24]. The resulting apurinic/apyrimidinic (AP) site is processed by AP-endonuclease 1 (APE1), allowing the downstream BER enzymes to complete the DNA repair process. Mutations in the human MYH (hMYH) gene can lead to colorectal cancer (as in MYH-associated polyposis or MAP) [25], while APE1 is essential for cell viability [26]. MYH contains unique motifs that mediate interactions with partner proteins involved in DNA replication, mismatch repair, and DDR (reviewed in [22], [23]). We have shown that the interdomain connector (IDC) located between the N- and C-terminal domains of hMYH is uniquely oriented [27] to interact with Hus1 [21] and APE1 [28].

The ring structure of 9–1–1 [29], [30], [31] is remarkably similar to that of the proliferating cell nuclear antigen (PCNA) [32], [33], [34]. Each 9–1–1 subunit folds into two globular domains linked by an interdomain connecting loop (IDCL) (Fig. 1A). According to the PCNA–DNA structure [35], the 9–1–1 ring is supposed to encircle double-stranded DNA [30]. Although the three subunits of the 9–1–1 complex are structurally similar, they exhibit key differences. These differences are most pronounced in the IDCLs between their N- and C-terminal domains [29], [30], [31]. These structural distinctions between 9–1–1 components have been suggested to dictate protein-binding specificity for individual subunits. For example, hMYH and hAPE1 bind preferentially to the Hus1 subunit [20], [21]. Functionally, the Hus1 subunit alone can stimulate MYH activity [21]. In this paper, we constructed a panel of Hus1 mutant proteins to identify domains required for binding MYH, stimulating MYH glycosylase activity, and binding DNA. Subsequently we tested the roles of other 9–1–1 components in MYH activation and DNA binding. This systematic and quantitative biochemical strategy revealed key differences in the ability of 9–1–1 subunits to bind DNA and functionally interact with MYH, thus supporting a model whereby each subunit of 9–1–1 plays a distinct and directed role in BER.