Structural Biochemistry/Nucleic Acid/DNA/9-1-1

Introduction
Because all living organisms are vulnerable to receiving damage to their DNA, cells have mechanisms which can activate a complex network of checkpoints and repair proteins so that the genetic information is unchanged and does not become mutated. One of these DNA repairing mechanisms is the 9-1-1 complex, which is a DNA-encircling, ring-shaped heterotrimer, is what is centrally responsible for damage control.

The 9-1-1 complex, which is an abbreviation for the RAD9-HUS1-RAD1 complex, is a close relative to the replication protein known as proliferating cell nuclear antigen (PCNA). When DNA damage is detected, the 9-1-1 complex is transported to the damaged DNA sites, where is serves as a platform to recruit both checkpoint and repair proteins. The interaction partners of the 9-1-1 complex have specific interaction boxes to interact with the complex, which allow them to distinguish between the 9-1-1 complex and the closely related PCNA. This discriminatory behavior of these interaction boxes enable even more specific interactions with the individual 9-1-1 subunits inside the 9-1-1 complex

Both endogenous and exogenous DNA damages are potentially harmful for an organism because it challenges the integrity of its genome. In order for the original genetic information of an organism to be preserved, errors in the DNA sequence must be able to be recognized and repaired by way of cellular mechanism. Cells have two highly interconnected mechanisms to achieve this goal. The first mechanism is a series of DNA damage checkpoints that detects and signals the presence of DNA damage, and the second mechanism is a DNA repair pathway which use an appropriate set of proteins that can successfully repair the specific DNA damage.

Comparison of PCNA and 9-1-1
Both PCNA and the 9-1-1 complex are two very structurally similar trimer ring-shaped complexes, which are also known as clamps. PCNA plays a vital role in DNA replication and replication-linked function, versus the 9-1-1 complex which plays a unique role in activation of checkpoints for DNA damage repair.

Although PCNA is only composed of one main unit, 9-1-1 actually has three subunits which differ between organisms; in Saccharomyces cerevisiae: Ddc1, Mec3, and Rad17, and in Homo sapiens: RAD9, HUS1 and RAD1, as the abbreviation of the complex suggests. It was not until recent studies by biochemists on the structure of the 9-1-1 complex that its role in DNA checkpoint signaling repair and meiosis has become more well-defined.

Similar to how the PCNA protein functions in DNA replication, the 9-1-1 complex encircles DNA specifically at its damaged sites, where it acts as a platform for checkpoint and DNA repair proteins to slow the progression of the cell cycle until the damage has been repaired. In addition, both PCNA and 9-1-1 complex use clamp loaders as their loading mechanisms. The two use different but related heteropentameric complexes as their loaders and constitute ATP-driven machines (AAA-ATPases) that can control whether or not the clamp remains open or closed and allow them to load onto DNA.

Despite their many similarities, PCNA and 9-1-1 do have their differences. PCNA is loaded to the 3' junctions of gapped DNA at the replication forks, whereas 9-1-1 is loaded onto the 5' junctions at the damaged DNA sites. This distinction is most likely brought by the state of the DNA and the behavior of the respective clamp loader complex. Also, PCNA has equivalent unit and subunit interfaces, but those in 9-1-1 are different.

Perhaps the most obvious structural difference between PCNA and 9-1-1 is that whereas PCNA only has one main unit, 9-1-1 is composed of three subunits that can be assembled in different combinations. However, recent data suggests that the only version that can be loaded onto damaged DNA and be functional in vivo is the heterotrimeric version of the ring. Moreover, despite similarities in the architecture between the PCNA protein and the 9-1-1 complex, structural studies revealed clear differences in the interdomain-connecting loops, which are important for binding for the PCNA protein. The three subnits that makes up the 9-1-1 complex also suggests that each of the subunits are specializations of functions and binding properties.

Functions
The main function of the 9-1-1 complex is to provide a platform for which DNA repair proteins can occur on damaged parts of DNA. After it binds to the damaged DNA sites, the DNA-bound 9-1-1 clamp initiates the activation of checkpoints and stops the cell cycle from progressing any further while the DNA remains damaged. This is the most important function of 9-1-1, as it serves as an early damage sensor and sets off a wave of protein-protein interactions to prevent genetic mutations.

Phosphorylation
More specifically, 9-1-1 triggers Sc-Mec1/Hs- ataxia telangiectasia and Rad3-related (ATR)-mediated phosphorylation and activation of Chk1 and Sc-Rad53/Hs-CHK2 protein kinases that conduct arrest in G1/S intra-S or G2-M phases of the cell cycle transcriptional upregulation of repair genes, downregulationa of cyclins and replication fork stabilization. In addition, yeast studies reveal that 9-1-1 cooperates with the cyclin-dependent kinase Sc-Cdc28 to recruit Sc-Dcd2 to DNA damage sites depending on the severity of the damage and the phase of the cell cycle.

DNA damage repair
9-1-1 further participates in repairing damaged DNA by binding to enzymes and factors of DNA repair pathways. The most notable example of which is base excision repair, which can be abbreviated as BER. The broad range of functions for 9-1-1 is further enhanced by its function in telomere regulation and apoptosis by recruiting the histone methyltransferase Set1 and the anti-apoptotic protein B-cell CLL/lymphoma 2, which can be shortened to BCL2.

Role in meiosis
9-1-1 also plays a very important role in meiosis by inducing the initiation of recombination between parental chromosomes as it is recruited to DNA double-strand breaks, also known as DSB's. Although this role is not a traditional role of the 9-1-1 complex, it is still equally vital to the cell.

Meiosis I is the stage at which genetic material of the parent is recombined and distributed to the two daughter cells. This process is started by the formation of the Spo11-induced DSB's at many genomic sites that are potentially lethal to the cell if not quickly repaired. The meiotic (pachytene) checkpoint plays a crucial role in controlling DSB processing and repair. This checkpoint pathway activates the central meiotic checkpoint kinase Mek1, as opposed to Rad53/CHK2, which play minor roles in meiosis, to prevent the progression of the meiotic cell cycle if the DSB's persist and coordinates recombination-associated events and guide meiotic progression. 9-1-1 becomes localized to chromosomes during the meiotic prophase, where it becomes phosphorylated. This process of the phosphorylation of 9-1-1 is dependent on the formation and processing of DSB's that are consistent with the known property of 9-1-1 to become loaded onto sites with damaged DNA.

The switch in checkpoint signaling can be achieved by a specific set of chromatin proteins. During meiosis, this specific set of chromatin proteins is expressed, which guides DSB repair in a way that encourages the recombination process between the parental chromosomes as opposed to the sister chromatids. Two of such chromatin proteins are Sc-Red1 and Sc-Hop1, which play two important roles during meiosis. First, they are the structural components of the synaptonemal complex and some of these often expressed proteins can also participate in the signaling process of meiotic checkpoints. Sc-Red1 interacts with two 9-1-1 subnits by way of two different PIP box-related motifs. More specifically, during meiosis, the Sc-Red1 protein becomes activated and acts as a direct downstream signaling partner of the 9-1-1 complex. Subsequently, this activation may lead to the phosphorylation of Sc-Hop1, as well as the activation of Mek1.

Yet another interesting phenomenon is that Sc-Red1 can bind to two different 9-1-1 subunits (Sc-Med3 and Sc-Ddc1), which protects these two subunits from interacting with Sc-Dpb11/Hs-TOPBP1. This competition model makes it so that the 9-1-1 complex that is bound to Sc-Red1 is unable to trigger the checkpoint signaling mechanism that is dependent upon Sc-Dpb11/Hs-TOPBP1. In contrast, it specifically induces the Hop1-Mek1-dependent checkpoint pathway, which is more commonly known as the meiotic checkpoint pathway. Though conventional checkpoint signaling mechanisms may still occur during meiosis, they are most likely mediated by 9-1-1 complexes which are not physically linked with Sc-Red1.

Regulations
Protein-protein interacts are altered by post-translational modifications. For instance, PCNA can be modified by ubiquitylation and SUMOylation, which can lead to the recruiting or repelling of binding factors. The ubiquitylation of PCNA is triggered by damaged DNA, which stalls replication. In turn, two different DNA damage tolerance pathways are facilitated. PCNA polyubiquitylaion gives an error-free repair pathways that has yet to be well understood. On the other hand, SUMOylation of yeast PCNA recruits the helicase Sc-Srs2, which has a SUMO-interacting motif. Because the yeast-specific residue is contained inside the interdomain-connecting loop of PCNA, it is suggested that the reversible SUMOylation at the site causes a reset by liberating all proteins that bind PCNA and give rise to the exchange of binding partners.

Given the similarities between PCNA and the 9-1-1 complex, it is thought that the modificiations by ubiquitin and SUMO may also be crucial for the regulation of 9-1-1 mediated events. Ubiquitylation of the Sc-Rad17 subunit has been shown to occur, but its function is still not yet clear.

In contrast, the role of 9-1-1 phosphorylation is much better understood by biochemists. The 9-1-1 subunit Sc-Ddc1/Hs-RAD9 has a flexible C-terminus tail which mediates the process of checkpoint signaling. By phosphorylating this tail, Sc-Dpb11/Hs-TOPBP1 is binded via the BRCT domains, which is a clear indicator that both the PCNA protein and the 9-1-1 complex utilize post-translational modifications to regulate the binding of co-factors.