Biochemistry DNA replication Essay Example

Biochemistry: DNA replication Paper Eukaryotic DNA replication is semi-discontinuous with the leading strand synthesized continuously and lagging strand is synthesized in fragments called Okazaki fragments (1). DNA replication starts at sites called replication origins present along the entire length of the chromosome (2). These sites have Origin of Replication Complex, which recruits different proteins to form replication fork (3). Helicases are one of those proteins that use energy from ATP hydrolysis to unwind the double stranded DNA to start the replication (4). Mcm2-7 proteins are considered to be a good candidate as helicases, as it has been shown to unwind the short double stranded DNA with 3’-5’ polarity (5). Mcm2-7 proteins have been co-immunoprecipitated with Cdc45 (6), which is an essential factor at the replication fork. GINS has also been shown to be necessary for the function of Cdc45 at the replication fork (7). Hypothesis: The role of Cdc45 in DNA replication is not well understood although it plays a critical part and has been shown to interact with various DNA replication machinery proteins such as Mcm2-7 and GINS. This study was designed to study the function of Cdc45 in Eukaryotic DNA Replication Summary of the results: 1. Purification and identification of the Cdc45/Mcm2-7/GINS (CMG) complex (Fig 1-2) a. Purification and co-immunoprecipitation of Cdc45 revealed 10 proteins as binding partners (Fig 1A-B). b. We will write a custom essay sample on Biochemistry: DNA replication specifically for you for only $16.38 $13.9/page Order now We will write a custom essay sample on Biochemistry: DNA replication specifically for you FOR ONLY $16.38 $13.9/page Hire Writer We will write a custom essay sample on Biochemistry: DNA replication specifically for you FOR ONLY $16.38 $13.9/page Hire Writer These are identified by mass spectroscopy and immunoblotting to be Mcm2-7 (6 proteins) and GINS complex (S1d5, Psf1, Psf2, and Psf3) proteins (Fig 1A, B). c. Cdc45 does not interact with individual Mcm proteins but only binds as a complex as it existed only in high molecular weight fractions (Fig 1C, Fig 2). d. These proteins form a stable, 11-member, high molecular weight complex. The mass of CMG complex is 700kDa as estimated through migration pattern in a Sepharose column. The calculated mass of one Mcm2-7 hexamer, one GINS tetramer and Cdc45 is 708kDa. So this CMG complex has only on Mcm2-7 hexamer and one GINS tetramer (Fig 1C, Fig 2). 2. Purification of the CMG complex to homogeneity (Fig 3A) a. The only way to purify intact CMG complex is by using an anti-Cdc45 affinity resin as other methods might disrupt the enzymatic activity of the complex. b. The complex was gently eluted using Cdc45 peptides that are hydrophilic and without secondary structure. c. This method of purification maintained the intrinsic enzymatic activity of the complex. 3. Identification of CMG complex for Helicase activity (Fig 3B – 3E) a. Only the complete 11-member CMG complex had helicase activity when assayed using a 40-bp duplex region with short tail annealed to a single stranded circle. b. Immunodepletion of Psf2 and Mcm5 proteins of the CMG using antisera against those proteins showed reduced helicase activity. This suggests that the activity is associated with the CMG complex and that Psf2 and Mcm5 are components of this complex. c. The various preparations of CMG complex also showed that the helicase activity is ATP dependent and is saturated at the concentration of 1mM ATP. 4. CMG directionality and processivity (Fig 4) a. Directionality assays using radiolabeled 5’ or 3’ primer with ssDNA in the middle revealed that CMG complex translocates on DNA in a 3’-5’ direction. b. The CMG complex can process substrates with a gapped single strand or 5’ or 3’ overhangs, just like the Mcm4, 6, 7 proteins was determined using a substrate with 5’ 30T tail and gapped single strand. c. The CMG complex can process many hundreds of base pairs at lower levels of protein compared to the Drosophila Mcm4, 6, 7 sub complex was established by using a population of duplex primer-plasmid regions of various heterogeneous lengths. 5. In Vivo Requirement of Cdc45 and GINS complex (Fig 5) a. RNA interference of Cdc45 or GINS complex proteins in Drosophila Kc tissue culture cells resulted in the accumulation of the cells in G1 and S phase of the cell cycle. This suggests that the cell cycle progression is impaired with the loss of Cdc45 or GINS member proteins. Conclusion: Cdc45, Mcm2-7 and GINS members form CMG complex, which forms the core of helicase activity for DNA replication. References: 1. Langston, LD. and O’Donnell, M. Mol. Cell 23: 155-160, 2006. 2. Maiorono, D. et al, Curr. Opin. Cell Biol. 18: 130-136, 2006. 3. Bell, SP. and Dutta, A. Annu. Rev. Biochem. 71: 333-374, 2002. 4. von Hippel, PH. And Delagoutte, E. BioEssays 25: 1168-1177, 2003. 5. You, Z. et al, Mol. Cell Biol. 19: 8003-8015, 1999. 6. Masuda, et al, Genes Cells 8: 145-161, 2003. 7. Takayama et al, Genes Dev. 17: 153-1165, 2003.

Six Frat Boys essays

Six Frat Boys essays The premise of this film is that there are six college aged students who represent six different stereotypes. There is a frat-boy type who attends every party on campus, a feminist activist who is offended by everything, computer-science major who spends his time making websites, a sweet, but somewhat flighty girl who is constantly being hit on, an armature rapper who makes up lyrics for everything he wants to say, and a wiccan girl who is constantly trying out new spells and incantations. These six students seem to have nothing in common with each other, but there is one central character that ties them all together. He is a slightly goofy student that makes extra cash by selling the answers to tests to students. Each of these students calls upon him and when he visits them they tell him their reasoning for needing his services instead of studying. As the plot unfolds and bits and pieces of the story are told, it turns out that each character is intertwined some way in the plot. Wh en each person is telling their version of the story, the other characters come off as being an exaggerated version of the stereotype they represent, to show how we sometimes classify people without getting to know them. The six people had all attended a party the previous weekend, without really knowing who each other were. The feminist character was attending the party to be able to write about the way women were treated at such events and when shes there she discovers the ditsy and decides to write about her and how misguided she is, but then ends up envying the attention she gets from all the males. The frat boy as a joke from his frat brothers follows the feminist around to try to mess with her, but ends up discovering she is not the man hating person everyone thought she was. The computer nerd was brought to the party by the rapper who wants him to make a website for his rap group and the computer guy ends up staying for the party and the two re...

Missions to Nicaragura Research Paper Example | Topics and Well Written Essays - 1500 words

Missions to Nicaragura - Research Paper Example Solving the problems faced by the people of Nicaragua as well as improving their life was the main aim of the mission groups. One of the mission groups is a BIMI mission group. The BIMI for Nicaragua is a Baptist international mission group that started its mission work in 1962. The mission group was started by Bob and Sabina Dayton. The main aim of BIMI mission group is to bring together and support missionaries and mission projects that were still going on in Nicaragua (BIMI, 2003). The group supported different mission groups in order for them to be in a position to serve better the people of Nicaragua. Another mission group is Nicaragua mission project which is a Christian movement mission that started in 1960s as a ministry to prisoners. The mission started with the name â€Å"the Christian mission in the jails†, and their founders were Rev Marceline Davila Castillo, a pastor in assemblies of God and Antonio Martinez who belonged to church of Nazarene. The group’s objective is to solve the problems faced by residents of this country by improving their life (Lubensky, 1999). The mission group goals were to build churches in order to spread the gospel and reach to people in all areas in that country. The group aimed at reinforcing ecumenical associations as well as promoting growth of churches as well as their communities. The group is a combination of various organizations such as CEPAD (Evangelical council of Nicaragua), Latin American council of churches, Disciples of Christ, united church of Christ and Christian Pentecostal church of Cuba. It has partnered with worldwide ministries for many years. The mission group started their mission by building churches, building bible schools for the people of Nicaragua and creating children’s programs (Baptist International Missions Inco, 2003). In its mission work, the missionary