The expression of the human Ki-67 protein is strictly associated with cell proliferation. During interphase, the antigen can be exclusively detected within the nucleus, whereas in mitosis most of the protein is relocated to the surface of the chromosomes. The fact that the Ki-67 protein is present during all active phases of the cell cycle (G(1), S, G(2), and mitosis), but is absent from resting cells (G(0)), makes it an excellent marker for determining the so-called growth fraction of a given cell population. In the first part of this study, the term proliferation marker is discussed and examples of the applications of anti-Ki-67 protein antibodies in diagnostics of human tumors are given. The fraction of Ki-67-positive tumor cells (the Ki-67 labeling index) is often correlated with the clinical course of the disease. The best-studied examples in this context are carcinomas of the prostate and the breast. For these types of tumors, the prognostic value for survival and tumor recurrence has repeatedly been proven in uni- and multivariate analysis. The preparation of new monoclonal antibodies that react with the Ki-67 equivalent protein from rodents now extends the use of the Ki-67 protein as a proliferation marker to laboratory animals that are routinely used in basic research. The second part of this review focuses on the biology of the Ki-67 protein. Our current knowledge of the Ki-67 gene and protein structure, mRNA splicing, expression, and cellular localization during the cell-division cycle is summarized and discussed. Although the Ki-67 protein is well characterized on the molecular level and extensively used as a proliferation marker, the functional significance still remains unclear. There are indications, however, that Ki-67 protein expression is an absolute requirement for progression through the cell-division cycle.

The Ki‐67 protein: From the known and the unknown

▪ Abstract The maintenance of the eukaryotic genome requires precisely coordinated replication of the entire genome each time a cell divides. To achieve this coordination, eukaryotic cells use an ordered series of steps to form several key protein assemblies at origins of replication. Recent studies have identified many of the protein components of these complexes and the time during the cell cycle they assemble at the origin. Interestingly, despite distinct differences in origin structure, the identity and order of assembly of eukaryotic replication factors is highly conserved across all species. This review describes our current understanding of these events and how they are coordinated with cell cycle progression. We focus on bringing together the results from different organisms to provide a coherent model of the events of initiation. We emphasize recent progress in determining the function of the different replication factors once they have been assembled at the origin.

DNA replication in eukaryotic cells

The p53 tumour-suppressor protein controls the expression of a gene encoding the p21 cyclin-dependent protein kinase (CDK) regulator. Levels of p21 protein are increased in senescent cells and p21 overexpression blocks the growth of tumour cells. In normal human cells, but not in many tumour cells, p21 exists in a quaternary complex with a cyclin, a CDK, and the proliferating-cell nuclear antigen (PCNA). p21 controls CDK activity, thereby affecting cell-cycle control, whereas PCNA functions in both DNA replication and repair. Here we use simian virus 40 DNA replication in vitro to show than p21 directly inhibits PCNA-dependent DNA replication in the absence of a cyclin/CDK. Furthermore, p21 blocks the ability of PCNA to activate DNA polymerase delta, the principal replicative DNA polymerase. This regulation results from a direct interaction between p21 and PCNA. Thus, during p53-mediated suppression of cell proliferation, p21 and PCNA may be important for coordinating cell-cycle progression, DNA replication and repair of damaged DNA.

The p21 inhibitor of cyclin-dependent kinases controls DNA replication by interaction with PCNA

Genome maintenance is a constant concern for cells, and a coordinated response to DNA damage is required to maintain cellular viability and prevent disease. The ataxia-telangiectasia mutated (ATM) and ATM and RAD3-related (ATR) protein kinases act as master regulators of the DNA-damage response by signalling to control cell-cycle transitions, DNA replication, DNA repair and apoptosis. Recent studies have provided new insights into the mechanisms that control ATR activation, have helped to explain the overlapping but non-redundant activities of ATR and ATM in DNA-damage signalling, and have clarified the crucial functions of ATR in maintaining genome integrity.

/pdf/atr-an-essential-regulator-of-genome-integrity-3i2zlw33w3.pdf

ATR: an essential regulator of genome integrity

Cellular DNA is subjected to continual attack, both by reactive species inside cells and by environmental agents. Toxic and mutagenic consequences are minimized by distinct pathways of repair, and 130 known human DNA repair genes are described here. Notable features presently include four enzymes that can remove uracil from DNA, seven recombination genes related to RAD51, and many recently discovered DNA polymerases that bypass damage, but only one system to remove the main DNA lesions induced by ultraviolet light. More human DNA repair genes will be found by comparison with model organisms and as common folds in three-dimensional protein structures are determined. Modulation of DNA repair should lead to clinical applications including improvement of radiotherapy and treatment with anticancer drugs and an advanced understanding of the cellular aging process.

https://science.sciencemag.org/content/sci/291/5507/1284.full.pdf

Human DNA Repair Genes

GINS maintains association of Cdc45 with MCM in replisome progression complexes at eukaryotic DNA replication forks

Little is known about the DNA helicases required for the elongation phase of eukaryotic chromosome replication. Minichromosome maintenance (MCM) protein complexes have DNA helicase activity but have only been functionally implicated in initiating DNA replication. Using an improved method for constructing conditional degron mutants, we show that depletion of MCMs after initiation irreversibly blocks the progression of replication forks in Saccharomyces cerevisiae. Like the Escherichia coli dnaB and SV40 T antigen helicases, therefore, the MCM complex is loaded at origins before initiation and is essential for elongation. Restricting MCM loading to the G1 phase ensures that initiation and elongation occur just once per cell cycle.

https://science.sciencemag.org/content/sci/288/5471/1643.full.pdf

Uninterrupted MCM2-7 Function Required for DNA Replication Fork Progression

Chromosome replication occurs precisely once during the cell cycle of almost all eukaryotic cells, and is a highly complex process that is still understood relatively poorly. Two conserved kinases called Cdc7 (cell division cycle 7) and cyclin-dependent kinase (CDK) are required to establish replication forks during the initiation of chromosome replication, and a key feature of this process is the activation of the replicative DNA helicase in situ at each origin of DNA replication. A series of recent studies has shed new light on the targets of Cdc7 and CDK, indicating that chromosome replication probably initiates by a fundamentally similar mechanism in all eukaryotes.

/pdf/how-do-cdc7-and-cyclin-dependent-kinases-trigger-the-4bmhehls69.pdf

How do Cdc7 and cyclin-dependent kinases trigger the initiation of chromosome replication in eukaryotic cells?

The accurate and complete replication of genomic DNA is essential for all life. In eukaryotic cells, the assembly of the multi-enzyme replisomes that perform replication is divided into stages that occur at distinct phases of the cell cycle. Replicative DNA helicases are loaded around origins of DNA replication exclusively during G1 phase. The loaded helicases are then activated during S phase and associate with the replicative DNA polymerases and other accessory proteins. The function of the resulting replisomes is monitored by checkpoint proteins that protect arrested replisomes and inhibit new initiation when replication is inhibited. The replisome also coordinates nucleosome disassembly, assembly, and the establishment of sister chromatid cohesion. Finally, when two replisomes converge they are disassembled. Studies in Saccharomyces cerevisiae have led the way in our understanding of these processes. Here, we review our increasingly molecular understanding of these events and their regulation.

/pdf/chromosome-duplication-in-saccharomyces-cerevisiae-1948h8f1q3.pdf

Chromosome Duplication in Saccharomyces cerevisiae.

Eukaryotic cells regulate the progression and integrity of DNA replication forks to maintain genomic stability and couple DNA synthesis to other processes. The budding yeast proteins Mrc1 and Tof1 associate with the putative MCM–Cdc45 helicase and limit progression of the replisome when nucleotides are depleted, and the checkpoint kinases Mec1 and Rad53 stabilize such stalled forks and prevent disassembly of the replisome. Forks also pause transiently during unperturbed chromosome replication, at sites where nonnucleosomal proteins bind DNA tightly. We describe a method for inducing prolonged pausing of forks at protein barriers assembled at unique sites on a yeast chromosome, allowing us to examine for the first time the effects of pausing upon replisome integrity. We show that paused forks maintain an intact replisome that contains Mrc1, Tof1, MCM–Cdc45, GINS, and DNA polymerases α and e and that recruits the Rrm3 helicase. Surprisingly, pausing does not require Mrc1, although Tof1 and Csm3 are both important. In addition, the integrity of the paused forks does not require Mec1, Rad53, or recombination. We also show that paused forks at analogous barriers in the rDNA are regulated similarly. These data indicate that paused and stalled eukaryotic replisomes resemble each other but are regulated differently.

/pdf/molecular-anatomy-and-regulation-of-a-stable-replisome-at-a-4ipfr328y3.pdf

Karim Labib

Papers

GINS maintains association of Cdc45 with MCM in replisome progression complexes at eukaryotic DNA replication forks

Uninterrupted MCM2-7 Function Required for DNA Replication Fork Progression

How do Cdc7 and cyclin-dependent kinases trigger the initiation of chromosome replication in eukaryotic cells?

Chromosome Duplication in Saccharomyces cerevisiae.

Molecular anatomy and regulation of a stable replisome at a paused eukaryotic DNA replication fork