Periodic Reporting for period 1 - Human Repl Mech (Mechanisms of human DNA replication)
Período documentado: 2019-04-01 hasta 2021-03-31
The eukaryotic DNA replication machinery consists of several dozen proteins. Initiation of DNA replication is a highly regulated process that ensures that the genome is duplicated exactly once per cell cycle. In G1 phase, the core of the replicative helicase, the Mcm2-7 complex (MCM) is loaded onto DNA at origins of replication. MCM loading depends on the origin recognition complex (ORC), Cdc6 and Cdt1. These proteins recruit MCM to DNA and load two MCMs onto DNA in the form of MCM double hexamers.
Helicase activation then happens in S phase, when the cell cycle-regulated kinases Dbf4-dependent kinase (DDK) and a cyclin-dependent kinase (CDK) become active. It involves the remodelling of MCM double hexamers to two active Cdc45-MCM-GINS (CMG) helicases, each encircling a single DNA strand. The remodelling involves several firing factors, phosphorylation and ATP hydrolysis. In budding yeast, DDK-phosphorylation of MCM recruits the firing factors Sld3/Sld7 (Treslin/MTBP in humans) and Cdc45. CDK-phosphorylation of Sld2 (RECQL4) and Sld3 (Treslin) promotes binding to Dpb11 (TOPBP1), which leads to recruitment of the tetrameric GINS complex and to CMG formation. Mcm10 and ATP hydrolysis then allow DNA unwinding and CMG activation. To form active replisomes additional proteins are recruited, such as the DNA polymerases Pol epsilon, Pol alpha-primase and Pol delta, PCNA and its loader the RFC complex, as well as Ctf4 (AND-1), Mrc1 (Claspin), Tof1 (Timeless) and Csm3 (TIPIN).
For the budding yeast proteins, an in vitro reconstitution system exists in which individual yeast DNA replication proteins can be purified and mixed together to recapitulate the processes of MCM loading, CMG activation and DNA replication in vitro. This system greatly facilitates studies of how DNA replication mechanisms work in yeast. While a clearer picture of the molecular details how the replicative helicase is loaded and activated in yeast is emerging, the steps leading to helicase loading and activation in mammals remain poorly understood. For example, the human MCM loading factors differ in complex composition and the human firing factors contain additional domains compared to their yeast orthologs. Furthermore, the phospho-regulation of the process might be different. Mechanistic studies are required to better understand how human DNA replication is initiated and how the process is regulated at the molecular level.
I am particularly interested in how the key replication initiation steps of MCM loading and CMG activation are mechanistically achieved and regulated in humans. Therefore, in this project I worked towards the following objectives:
(I) The first objective is to reconstitute the human DNA replication machinery using recombinant human proteins expressed in baculovirus-infected insect cells. I am combining methodology that I developed during my PhD for the rapid generation of human protein complexes with the host lab's expertise in the reconstituted yeast replication system.
(II) The second objective is to use the reconstitution system to study mechanistic details of human replication initiation.
The reconstitution system will facilitate mechanistic studies of human DNA replication and could in future enable studies on disease mechanisms. Defects in the replication machinery are involved in a range of diseases such as Meier-Gorlin syndrome and the process is misregulated in cancer.
Helicase activation then happens in S phase, when the cell cycle-regulated kinases Dbf4-dependent kinase (DDK) and a cyclin-dependent kinase (CDK) become active. It involves the remodelling of MCM double hexamers to two active Cdc45-MCM-GINS (CMG) helicases, each encircling a single DNA strand. The remodelling involves several firing factors, phosphorylation and ATP hydrolysis. In budding yeast, DDK-phosphorylation of MCM recruits the firing factors Sld3/Sld7 (Treslin/MTBP in humans) and Cdc45. CDK-phosphorylation of Sld2 (RECQL4) and Sld3 (Treslin) promotes binding to Dpb11 (TOPBP1), which leads to recruitment of the tetrameric GINS complex and to CMG formation. Mcm10 and ATP hydrolysis then allow DNA unwinding and CMG activation. To form active replisomes additional proteins are recruited, such as the DNA polymerases Pol epsilon, Pol alpha-primase and Pol delta, PCNA and its loader the RFC complex, as well as Ctf4 (AND-1), Mrc1 (Claspin), Tof1 (Timeless) and Csm3 (TIPIN).
For the budding yeast proteins, an in vitro reconstitution system exists in which individual yeast DNA replication proteins can be purified and mixed together to recapitulate the processes of MCM loading, CMG activation and DNA replication in vitro. This system greatly facilitates studies of how DNA replication mechanisms work in yeast. While a clearer picture of the molecular details how the replicative helicase is loaded and activated in yeast is emerging, the steps leading to helicase loading and activation in mammals remain poorly understood. For example, the human MCM loading factors differ in complex composition and the human firing factors contain additional domains compared to their yeast orthologs. Furthermore, the phospho-regulation of the process might be different. Mechanistic studies are required to better understand how human DNA replication is initiated and how the process is regulated at the molecular level.
I am particularly interested in how the key replication initiation steps of MCM loading and CMG activation are mechanistically achieved and regulated in humans. Therefore, in this project I worked towards the following objectives:
(I) The first objective is to reconstitute the human DNA replication machinery using recombinant human proteins expressed in baculovirus-infected insect cells. I am combining methodology that I developed during my PhD for the rapid generation of human protein complexes with the host lab's expertise in the reconstituted yeast replication system.
(II) The second objective is to use the reconstitution system to study mechanistic details of human replication initiation.
The reconstitution system will facilitate mechanistic studies of human DNA replication and could in future enable studies on disease mechanisms. Defects in the replication machinery are involved in a range of diseases such as Meier-Gorlin syndrome and the process is misregulated in cancer.
With the aim of establishing an in vitro reconstitution system of human DNA replication, proteins were expressed using the baculovirus-insect cell expression system. For this, human cDNA was generated after RNA isolation from human cells. Then, I generated baculoviral expression vectors for individual replication proteins and for multisubunit protein complexes using the biGBac vector system. All expression constructs were sequence verified and proteins were expressed in insect cells.
The first step in replication initiation is the loading of the core of the replicative helicase, MCM, onto DNA in a process that depends on ORC, Cdc6, Cdt1 and ATP. In contrast to the yeast proteins, human ORC6 does not copurify with ORC1-5. Another difference to the yeast system is that human CDT1 does not form a stable complex with MCM2-7. I was however able to purify the complexes ORC1-5 and MCM2-7 as well as the individual proteins ORC6, CDT1 and CDC6. I developed DNA recruitment assays, in which biotinylated DNA is immobilized on Streptavidin-coated beads. Results indicate that human ORC can be recruited to these DNA beads. Recruitment of human MCM to DNA beads depended on ORC and on ATP.
The second step in replication initiation is helicase activation. This involves the action of kinases and firing factors and leads to the formation Cdc45-MCM-GINS (CMG) helicases. I expressed and purified human CDC45, tetrameric GINS complex, as well as the firing factors Treslin-MTBP, RECQL4, TOPBP1 and the kinases Dbf4-dependent kinase (DDK) and cyclin-dependent kinases (CDK). Further DNA replication proteins that I expressed and purified included the human DNA polymerases Pol epsilon, Pol alpha-primase, Pol delta, replication protein A (RPA), replication factor C (RFC), Timeless/TIPIN, Claspin and AND-1. Most human replication proteins could be purified using this system, while some require further optimization.
I am using the generated human DNA replication proteins for my aim to establish an in vitro reconstitution system of human DNA replication that can recapitulate the steps of MCM loading, CMG activation, and DNA replication in vitro. I am using parts of the system to study mechanisms of human replication initiation. I expect the reconstitution system to become a valuable resource for the study of human DNA replication mechanisms. The system and the results of my study will be published and made available to the scientific community.
The first step in replication initiation is the loading of the core of the replicative helicase, MCM, onto DNA in a process that depends on ORC, Cdc6, Cdt1 and ATP. In contrast to the yeast proteins, human ORC6 does not copurify with ORC1-5. Another difference to the yeast system is that human CDT1 does not form a stable complex with MCM2-7. I was however able to purify the complexes ORC1-5 and MCM2-7 as well as the individual proteins ORC6, CDT1 and CDC6. I developed DNA recruitment assays, in which biotinylated DNA is immobilized on Streptavidin-coated beads. Results indicate that human ORC can be recruited to these DNA beads. Recruitment of human MCM to DNA beads depended on ORC and on ATP.
The second step in replication initiation is helicase activation. This involves the action of kinases and firing factors and leads to the formation Cdc45-MCM-GINS (CMG) helicases. I expressed and purified human CDC45, tetrameric GINS complex, as well as the firing factors Treslin-MTBP, RECQL4, TOPBP1 and the kinases Dbf4-dependent kinase (DDK) and cyclin-dependent kinases (CDK). Further DNA replication proteins that I expressed and purified included the human DNA polymerases Pol epsilon, Pol alpha-primase, Pol delta, replication protein A (RPA), replication factor C (RFC), Timeless/TIPIN, Claspin and AND-1. Most human replication proteins could be purified using this system, while some require further optimization.
I am using the generated human DNA replication proteins for my aim to establish an in vitro reconstitution system of human DNA replication that can recapitulate the steps of MCM loading, CMG activation, and DNA replication in vitro. I am using parts of the system to study mechanisms of human replication initiation. I expect the reconstitution system to become a valuable resource for the study of human DNA replication mechanisms. The system and the results of my study will be published and made available to the scientific community.
At present, an in vitro reconstitution system of eukaryotic DNA replication that is able to recapitulate the steps of MCM loading, CMG activation and DNA replication exists only for budding yeast proteins. Development of a reconstitution system of human DNA replication will facilitate and enable studies on human DNA replication mechanisms. A range of human diseases are associated with defects in the DNA replication machinery or with its misregulation. The reconstitution system could allow studies on disease mechanisms and could facilitate drug discovery.